6alpha-methyl-21 halo nor ethisterones and intermediates in the preparation thereof



United States Patent 3,211,725 6a-METHYL-21 HALO NOR ETHISTERONES AND INTERMEDIATES IN THE PREPARATION THEREOF Arthur E. Oberster, Canton, Ohio, Roger E. Beyler, Carbondale, Ill., and Lewis H. Sarett, Princeton, N.J., assignors to Merck & Co., Inc., Rahway, N.J., a corporation of New Jersey No Drawing. Filed Apr. 20, 1960, Ser. No. 23,396 10 Claims. (Cl. 260239.55)

This invention is concerned generally with novel steroids and processes of preparing the same. More particularly, it relates to novel 2l-haloethisterones, 21-halonorethisterones, and closely related compounds which possess useful therapeutic properties as orally and parenterally active progestational agents. This invention also relates to pharmaceutical compositions containing ,these novel steroid compounds.

In accordance with the present invention, the novel 21- haloethisterones (17u-haloethynyl-4-androstene-1713-01-3- ones) and the 21-halonorethisterones (17a-halo-ethynyll9-nor-l7fl-ol-3-ones) are prepared from a 17a-ethynyl- 3-oxygenated-androstene-17,8-01 by first protecting the oxygen functions present at the C-3 and C-17 positions of the steroid molecule with suitable protecting groups, and then halogenating to form the 17a-haolethynyl derivative. The protecting groups at C-3 and C-17 are then removed.

This process may be schematically represented as follows, starting with a 17a-ethynyl-4-androstene-1713-01-3- one in which R is methyl, or the corresponding 19-n0rsteroid in which R is hydrogen:

HO CECE HO GECH Step 1. 0 o: O

I IIA O o pzon o CECX Step 2. Step 3. O O v s O t 0 I IIB III HO\ CEOX HO\ CECX IV-B IV-A

3,211,725 Patented Oct. 1 2 1965 no czox IV-C When starting with the 19-nor-l7u-ethynyl-4-androstene-17fl-o1-3-one, in which R is hydrogen, Compound II-A (and likewise Compounds H- B and III) are mixtures of the A and the A -andr0stenes, and in the case of the A compound, the hydrogen (R) shown at (3- 10 is present at C-4. Removal of the protecting groups in Step 3, using concentrated HCl gives only the 3-keto-A -androstene (IV-A), but using approximately 70% aqueous acetic acid gives a mixture of the 3 -keto-A (IV-A), the 3-keto-A -(IVB) and the 3-keto-A -androstenes, which compounds can be separated by chromatography.

When starting with the 17a-ethynyl-4-androstenel'7fi-ol-3-one, in which R is methyl, only the A -androstene is present at II-A, II-B and III, and only the A androstene (IV-A) is formed in Step 3.

The process may be schematically represented in the following manner, starting with a 17a-ethynyl-5-androstene-36,17fl-diol in which R is methyl, or the corresponding 19-nor-steroid in which R is hydrogen:

HO CECH If the starting material is the l7-a-ethynyl-5-androstene-3/3,17/3-diol (I'), the product (IV) which is obtained on removal of the protecting groups is the 170:- haloethynyl-S-androstene 35,17,8-diol. This compound may be converted into the 17a-haloethynyl-4-androstene- 17fl-o1-3-one (21-haloethisterone) by oxidation, for example, with aluminum isopropoxide and cyclohexanone (or acetone) in benzene. The choice of the 3-oxygenatedsteroid used as starting material is based on practical considerations, availability and the like.

In the first step of our process the oxygen functions present at the C-3 and C-l7 positions of the steroid molecule are protected. In a preferred embodiment of our invention a 3-keto group is blocked by forming the 3- ethylenedioxy-derivative by reaction with excess ethylene glycol in benzene, toluene or ethylene dichloride solvent in the presence of acid catalysts such as p-toluenesulfonic or sulfuric acid, the water by-product being continuously removed.

The 3-keto group may also be converted into the 3- ethylenedioxy derivative by exchange dioxolanation, which involves acid-catalyzed transfer of the ethylene glycol portion of simple 2,2-dialkyl-l,3-dioxolanes, such as 2,2-dimethy1-1,3-dioxolane (acetone ethylene ketal) or, better, 2-methyl-2-ethyl-1,3-di0xolane (butanone ethylene ketal), on reaction of the ketones either in an inert solvent, as benzene, or simply in excess reagent.

Other cyclic ketal derivatives can be used in our process for protecting a ketone group at C-3. In general, we have found that the lower alkylenedioxy derivatives wherein the hydrocarbon group contains not more than seven carbon atoms, such as the ethylenedioxy, trimethylenedioxy, propylenedioxy and butylenedioxy derivatives are most suitable. However, in place of using a lower alkylenedioxy substituent to block or protect the keto substituent, we can use other derivatives readily hydrolyzable to keto, such as an enol ether monothioketal, or a dithioketal derivative for this purpose.

The 17,8-01 group, and the 3B,17 8-dio1 groups in the steroid molecule are protected by reaction with dihydropyran in the presence of an acidic reagent, such as ptoluenesulfonyl chloride, to form the corresponding tetrahydropyranyl ether. This reaction takes place, for example, on addition of p-toluenesulfonyl chloride to a solution of the steroid in an excess of dihydropyran. The reaction mixture is stirred at room temperature for 16 to 64 hours. The product may be recovered by neutralizing the reaction mixture with dilute base, and then extracting with ether. The ether extracts are washed with water, dried and evaporated to dryness. The crude product is dissolved in a solvent such as petroleum ether, and chromatographed over alumina to give the corresponding tetrahydropyranyl derivative.

In Step 2 of our invention, the steroid starting material, protected at the 3- and the 17-positions as indicated above, is treated with a suitable halogenating agent. In the preferred embodiment of our invention, the steroid compound, dissolved in a tertiary alcohol, is first treated with a potassium alcoholate of that tertiary alcohol, to form the 2'l-potassium derivative of the steroid compound, and the latter compound is then halogenated.

To form the 21-chloro-derivative, the steroid is dissolved in t-butyl alcohol and treated with potassium-tbutoxide and then with t-butyl-hypochlorite.

To form the ZI-bromo-derivative, the steroid is dissolved in t-butyl alcohol and treated with potassium tbutoxide and then with N-bromosuccinimide. The choice of the tertiary alcohol reagents is determined by practical considerations, such as availability, solubility and the like.

The 2l-halo-steroid compounds are conveniently recovered by adding water to the reaction mixture, and extracting with ether. The ether extracts are then washed with water, dried, and evaporated to dryness in vacuo. The residual material thus obtained is separated by chromatography on alumina to afiord the 2-1-halo-steroid.

In Step 3 of our process, the protecting groups at C-3 and 0-17 are removed, preferably by treatment with acid. Removal of the protecting groups using concentrated HCl gives the corresponding 3-keto-A -androstene. To carry out this reaction, a solution of the steroid in methanol is stirred with concentrated HCl for approximately one hour at room temperature. The methanol may then be removed under reduced pressure. The steroid is conveniently recovered by ether extraction. The ether extract is dried, evaporated to dryness and the residual material crystallized from a suitable solvent.

When starting with the 19-nor-17a-ethynyl-4-androstene-17B-ol-3-one, in which R is hydrogen, removal of the protecting groups from the mixture of the 19-nor-A and A -androstenes present in III by treatment with approximately aqueous acetic acid for about one hour at room temperature gives a mixture of the 3-keto- 19-nor-A the 3-keto-l9-nor-A and the 3-keto-19- nor-A androstenes. These compounds can be separated by chromatography over acid-washed alumina and elution with mixtures of ether and petroleum ether.

The 170: haloethynyl-S-androstene-3,8,17,8-diol (IV) may be converted into the 17a-haloethynyl-4-androstene- 17fi-ol-3-one (ZI-haloethisterone) (V) by oxidation with aluminum isopropoxide and cyclohexanone in benzene. This oxidation is carried out by dissolving the steroid in a solvent such as a mixture of cyclohexanone and benzene and reacting in an inert atmosphere with a solution of aluminum isopropoxide in benzene at -90 C. for 2-16 hours. To recover the product, the solution is cooled, a few drops of water are added, and the resulting aluminum hydroxide is filtered off. The filtrate is concentrated on the steam bath under reduced pressure, and the residual material is crystallized from a suitable solvent.

In accordance with the present invention, the novel 21- haloethisterones are prepared from the 17a-et-hynyl-5- androstene-3p,17B-diol by first reacting the latter compound with dihydropyran in the presence of p-toluenesulfonyl chloride to give the 17a-ethynyl-5-androstene-3B, l7 3-diol-bis-tetrahydropyranyl ether.

The 17a-ethynyl-5-androstene=3,8,17 3-diol-bis-tetrahydropyranyl ether is chlorinated by dissolving in t-butyl alcohol and treating with a solution of potassium t-butoxide and then with t-butyl hypochlorite to give the chloroethynyl-S-androstem-313,17fl-diol-bis-tetrahydropyranyl ether. The latter compound is then hydrolyzed with concentrated HCl to give I'I'u-ChIOI'O ethylnyl-5-androstene-3/3,17,8-diol, which is then oxidized; with aluminum isopropoxide and cyclohexanone to give; the 17m chloroethynyl 4-androstene-17fl-ol-3-one (21- chloroethisterone).

The 17a-ethynyl-5-androstene-3p,17/3 diol-bis-tetrahy-- dropyranyl ether is brominated by dissolving the steroid;

in t-butyl alcohol and treating with a solution of potassium t-butoxide and then with N-bromosuccinimide to give the 17ot-bromoethynyl-5-androstene-3fi,17,8-diol-bistetrahydropyranyl ether. The latter compound is then hydrolyzed with concentrated HCl to give l7a-bromioethynyl-5-androstene-3fl,17,8-diol, which is then oxidized with aluminum isopropoxide and cyclohexanone in benzene to give the 17a-bromoethynyl-4-androstene-l75-01-3- one (21-bromo-ethisterone) The novel 2l-halonorethisterones are prepared from norethisterone (l7u-ethynyl-l9-nor-4-androstene-17,8-01- 3-one) by reaction first with ethylene glycol in the presence of p-toluenesulfonic acid to give a mixture of the A and the A -17a-ethynyl-3-ethylenedioxy-19 norandrostene-lm-ol, and then with dihydropyran to give the corresponding tetrahydropyranyl ether.

The mixture of the A and the A -17o-ethynyl-3- ethylenedioxy-l9-nor-androstene-l7fi-ol tetrahydropyranyl ether is chlorinated by dissolving the steroid in t-butyl alcohol and treating with a solution of potassium t-butoxide and then with t-butylhypochlorite to give a mixture of the A and the A -lh-chloroethynyl-19- nor androstene l7p3-ol-3-one tetrahydropyranyl ether, which mixture is then hydrolyzed with acid. Hydnolysis of the mixture with concentrated HCl gives the l7oz-Cl1l0- roethynyl-l9-nor-4-androstene-17 8-ol-3-one (2l-chloro norethisterone). Hydrolysis of the mixture with 70% aqueous acetic acid gives a mixture of the M, the A and the A -l7a-chloroethynyl-19-nor-androstene-17,6-01-3- ones, which mixture is separated by chromatography.

The mixture of the A and the A -17u-ethynyl-3- ethylenedioxy 19 nor androstene-17B ol tetrahydropyranyl ether is brominated by dissolving the steroid in t-butyl alcohol and treating with a solution of potassium t-butoxide and then with N-bromosuccinimide to give a mixture of the A and the A -l7a-bromoethynyl-l9 nor androstene l7,B-ol-3-one tetrahydr-opyranyl ether. The latter compound is then hydrolyzed with acid. Hydrolysis of the mixture with concentrated HCl gives the 17a-bromoethynyl-19-nor-4-androstene-l7p-ol-3-one (21- bromonorethistenone). Hydrolysis of the mixture with 70% aqueous acetic acid gives a mixture of the A The A and the A -l7a-bromoethynyl-19-nor-androstene- 17/3-ol-3-ones, which mixture is separated by chromatography.

In addition to the above described 17u-haloethynyl-4- androstene-l7fi-ol-3 ones, this invention contemplates the preparation of certain derivatives thereof, and in particular the novel steroid compounds which may be chemically represented as follows:

OR X

wherein the 1,2-positions and the 6,7-positions of the steroid molecule may be double bonded or saturated, and wherein R is hydrogen, a methyl group, R is a lower alkyl or alkanoyl group, X is CECCI, CECBI, CHzCHCl CH=CHBr, CH CH Br or CH CH CI, and Y is methyl, chloro or fluoro.

This invention also contemplates the preparation of the 17,8-lower alkyl esters and ethers of the l7a-haloethynyl- 19-nor-5(l0)-androstene-17fi-ol 3 ones, as well as the products which are formed on hydrogenating the 170:- haloethynyl group of the aforesaid compounds to the 17a-CH=CHX, and to the 17a-CH CH X groups, where X represents chlorine or bromine.

6 The l7a-haloethynyl group of the 21-haloethisterones and the 2l-halonorethisterones (VI) is reduced to the corresponding chlorovinyl derivative (VI) on hydrogenation with a Lindlar catalyst, and to the corresponding chloroethyl derivative (VII) on hydrogenation using a heavy metal catalyst such as PtO 'CECX The 6a-methyl-2l-haloethisterone is prepared by reacting the 17a-haloethynyl-5-androstene-3[3,17,8-diol with monoperphthalic acid to give the 17a-haloethynyl-androstane-3B,17,8-diol-5,6-oxide, which on reaction with methyl magnesium iodide yields 6fi-methyl-17a-haloethynyl-androstane-3 9,5a,17fl-triol. The latter compound is oxidized with an oxidizing reagent prepared from chromium trioxide and sulfuric acid to give 6B-methyl-17a-haloethynyl-androstane-Sa,17B-diol-3-one, which on treatment with aqueous sodium hydroxide gives the 6a-methyl-17ahaloethynyl-4-androstene-l7fi-ol-3-one.

The 6a-methyl-17a-haloethynyl-4-androstene-173-01-3- one is dehydrogenated at C-1 by means of selenium dioxide, or alternately by microbiological methods, to give the Ga-methyl-17a-haloethynyl-1,4-androstadiene -01- 3-one.

The 6a-methyl-l7u-haloethynyl-4-androstene-1713-01-3- one is reacted with chloranil to give 6-methyl-l7oc-haloethynyl-4,6-androstadiene-17B-ol-3-one, which compound is dehydrogenated at C4, by means of selenium dioxide, or alternately by microbiological methods, to give 6- methyl-l7a-haloethynyl-1,4,6-androstatriene-17B-ol-3-one.

The 17(3-1ower alkyl ethers of the 17B-hy-droxy-17ahaloethynyl-3-keto-steroids are prepared by the reaction of the corresponding l7fl-hydroxy-steroid with a lower alkyl halide and silver oxide in a solvent such as di-methylformamide. The lower alkyl halides which may be used for this purpose include methyl iodide, ethyl iodide, n-propyl iodide, n-butyl iodide and the like.

The 17,8-lower alkanoyl ethers of the l7B-hydroxy-l7ahaloethynyl-3-ketosteroids are prepared by the reaction of the corresponding 17/3-hydroxy-steroid with a lower alkanoic acid anhyd-ride in the presence of an organic base such as pyridine. The lower acid anhydrides which may be used for this purpose include acetic anhydride, propionic anhydride, butyn'c anhydride, and the like.

The l7u-haloethynyl-1,4-androsta-diene-175-01 3 one acetate is prepared by reacting 17a-haloethynyl-4-androstane 17fl-ol-3-one with acetic anhydride in pyridine to give the corresponding acetate, and then converting the latter compound into the 17a-haloethynyl-1,4-androstadiene-17fl-ol-3-one acetate by means of selenium dioxide, or alternately by microbiological methods.

The l7ut-haloethynyl-1,4 androstadiene-175-methoxy- 3-one is prepared by reacting 17a-haloethynyl-4-androstene-17fl-ol-3-one with methyl iodide and silver oxide, using dimethyl-formamide as solvent, to give the 17mhaloethynyl-4-androstene-17,8-methoxy 3 one and then converting the latter compound into the 17a-haloethynyl- 1,4-androstadiene-l7fi-methoxy-3-one by means of selenium dioxide, or alternately by microbiological methods.

The 6a-chloro-17u-haloethynyl-1,4-androstadiene-175- ol-3-one acetate is prepared by reacting 17a-hal0ethynyl- 4-androstene-17fi-ol-3-one with acetic anhydride and ptoluenesulfonic acid to give the 17a-haloethynyl-3,5-androstadiene-3,17/i-diol-diacetate. The latter compound is reacted with N-chlorosuccinimide to give the 60t-Ch1OIO- 17a-haloethynyl-4-androstene-17/3-01-3-one acetate, which is converted into the 6a-chloro-17a-haloethynyl-1,4-androstadiene-17/3-ol-3-one acetate by reaction with selenium dioxide, or alternately by microbiological methods.

The 6u-chloro-17a-haloethynyl-1,4,-androstadiene-175- methoxy-B-one is prepared by reacting l7a-haloethynyl- 4-andostene-17B-ol-3-one with methyl iodide and silver oxide, using dimethylformamide as solvent, to give the 17m haloethynyl-4-androstene-17/3-methoxy-3-one. The latter compound is then converted into the 17oc-hal0- ethynyl-3,5-androstadiene-17,8-methoxy-3-ol acetate, by reaction with acetic acid and p-toluenesulfonic acid. The acetate so formed is treated with N-chlorosuccinimide to give the 6a-chloro-17u-haloethynyl-4-androstene-175- methoxy-Bone, which compound is dehydrogenated by selenium dioxide, or alternately by microbiological methods, to the 6u-chloro-17a-haloethynyl-1,4-androstadiene- 17/3-methoxy-3-one.

The 6 chloro-17a-haloethynyl-4,6-androstadiene-17B- ol-3-one acetate is prepared by reaction of 17a-halo ethynyl-4-androstene-l7 3-ol-3-one with chloranil to give the 17a-haloethynyl-4,G-androstadiene-17 8-ol-3-one (A 21-haloethisterone), which is converted into the 17a-haloethynyl-4-androstene-17fl-ol-3-one-6,7-oxide on reaction with perbenzoic acid. The latter compound is reacted with HCl in chloroform solution to give the 6-chlor0 l7ahaloethynyl-4,6-androstadiene-17,8-01-3-one which forms the acetate on treatment with acetic anhydride in pyridine, and the corresponding l7/3-methoxy-derivative on reaction with methyl iodide and silver oxide. The 6-Cl1lOIO-17ochaloethynyl-4,6-androstadiene-17,B-ol-3-one acetate is dehydrogenated to the 6-chloro-l7a-haloethynyl-1,4,6-androstatrienl-l7/3-01-3-one acetate on reaction with selenium dioxide, or alternately by microbiological methods.

The 6a fluoro l7ot-haloethynyl-4-androstene-1713-01- 3-one acetate is prepared from the 17u-haloethynyl-3,5-androstadiene-3,17B-diol diacetate by reaction with perchloryl fluoride followed by acid treatment. The 6ozfluoro-17u-halocthynyl-4-androstene-l7 6-ol-3-one acetate is dehydrogenated at C-1 by means of selenium dioxide, or alternately by microbiological methods, to give 60:- fluoro 170a haloethynyl-1,4-androstadiene-17,8-01-3-one acetate.

The 6a-fluoro-17a-haloethynyl-4-androstene-17,8-ol-3- one acetate is reacted with chloranil to give 6-fluoro-17ahaloethynyl-4,6-androstadiene-l7fl-ol-3-one acetate, and the latter compound is dehydrogenated at C1 by means of selenium dioxide, or alternately by microbiological methods, to give 6-fiuoro-l7tx-haloethynyl-l,4,6-androstatriene-17fl-ol-3-one acetate.

The 17a-haloethynyl-3,5-androstadiene-17fi-methoxy- 313-01 acetate is reacted with perchloryl fluoride followed by acid treatment to give 6a-fiuoro-17a-haloethynyl-4- androstene-17fl-methoxy-3-one, which compound may be dehydrogenated at C-1, by means of selenium dioxide, or alternately by microbiological methods, to give 6a-fiuoro- 17a-haloethynyl-l,4-androstadiene-l7,8-methoxy-3-one.

The 6a-fluoro-l7a-haloethynyl-4-androstene-17,8-methoxy-3-0ne is converted into 6-fluoro-17a-haloethynyl-4,6- androstadiene-17fl-methoxy-3-one by the use of chloranil. The 6 fluoro 17a-haloethynyl-4,6-androstadiene-17/3- methoxy-3-one is dehydrogenated at C-1 by means of selenium dioxide, or alternately by microbiological methods, to yield 6-fluoro-17a-haloethynyl-1,4,6-androstatricue-17,8-methoxy-3-one.

The 17a-haloethynyl-4-androstene-17fi-ol-3-one is reduced to 21-halo-4,20-pregnadiene-17fl-ol-3-one by hydrogenation in ethyl acetate, using a Lindlar catalyst, and to 21-halo-4-pregnene-17/9-ol-3-one by hydrogenation in ethyl alcohol using a platinum oxide catalyst.

The 6a-methyl-derivatives of the 21-halonorethisterones are prepared by first converting the 21-halonorethisterones (17u-haloethynyl-19-nor-4-androstene-l7,8-ol-3-one) into the 17a-haloethynyl-19-n0r-5-androstene-3/i,17/3-diol-l7fiacetate by reacting the l7a-haloethynyl-19-nor-4-androstene-17/3-ol-3-one with acetic anhydride and p-toluene sulfonic acid to give the l7a-haloethynyl-19-nor-3,5-androstadiene-3,17/8-diol-diacetate and then reacting the latter compound with sodium borohydride. Oxidation of 17a haloethynyl 19-nor-S-androstene-3B-17fi-diol-17l3- acetate with monoperphthalic acid yields 17a-haloethynyll9 nor androstane-3fi, 17B-diol-5,6-oxide-17fi-acetate. The latter compound is reacted with methyl magnesium iodide to form 6/3-methyl-17u-haloetl1ynyl-19-nor-androstane-3/3,5a,17B-triol, which on oxidation with chromic oxide in acetone gives 6B-methyl-17a-haloethynyl-l9-norandrostane-Sa,17,8-diol-3-one. The latter compound is treated with sodium hydroxide to form 6a-methyl-17ahaloethynyl-19-nor-4-androstene-l7B-0l-3-one.

The 6a-methyl-17a-haloethynyl-19-nor-4 androstene- 17/3-ol-3-one is converted to 6a-methyl-17u-haloethynyl- 19-nor-4-androstene-17B-methoxy-3-one by reaction with methyl iodide and silver oxide. The 6a-methyl-17ahaloethyl-4-androstene-l7/3-ol-3-one is treated with acetic anhydride in pyridine to give 6ot-methyl-17-haloethynyl-l9- nor-4-androstene-17;3-ol-3-one acetate.

The 60: chloro-17oa-haloethynyl-19-nor-4-androstene- 17fi-0l-3-one acetate is prepared from the l7a-haloethynyl-19-nor-3,5-androstadiene-3,l7fi-diol diacetate by reacting with N-chlorosuccinimide.

The 6a-fiuoro-17a-haloethynyl 19 nor-4-androstene- 17/3-ol-3-one acetate is prepared from the 17a-hal0- ethynyl 19 nor-3,5-androstadiene-3,17,8-diol diacetate by reacting with perchloryl fluoride followed by acid treatment.

The 6a-chloro-l7a-haloethynyl l9 nor-4-androstene- 17,6-methoxy-3-one is prepared from the 17a-haloethynyl-19-nor-4-androstene-17/i-ol-3-one by reacting first with silver oxide and methyl iodide to form the haloethynyl-19-nor-4-androstene-17fi-methoxy 3 one. The latter compound is then reacted with acetic anhydride and p-toluenesulfonic acid to give the 17u-haloethynyl-l9-nor-3,5-androstadiene -17;8 methoxy 3-01 acetate, which is converted into the 6a-ChlO10-17oc-hal0- ethynyl-19-nor-4-androstene-17fi methoxy 3 one on treatment with N-chlorosuccinimide.

The 6a-fiu0ro-17u-ha1oethynyl 19 nor-4-androstene- 17/3-methoxy-3-one is obtained by reacting the 17a-haloethynyl 19-nor-3,5-androstadiene-17/3-methoxy-3-ol acetate with perchloryl fluoride followed by acid treatment.

The 17a-haloethynyl 19 nor-4-androstene-17fi-ol- 3-one is reduced to 2l-halo-19-nor-4,20-pregnadiene-17,8- ol-3-one by hydrogenation in ethyl acetate, using a Lindlar catalyst and to 2l-halo-19-nor-4-pregnene-175-01- 3-one by hydrogenation in ethyl alcohol, using a platinum oxide catalyst.

The 17a-haloethynyl 19 nor (10)-androstene-17flol-3-one forms the acetate on treatment with acetic anhydride in pyridine, and the corresponding 17,8-methoxyderivative on reaction with methyl iodide and silver oxide. The 17u-haloethynyl 19 nor-5(l0)-androstene 17B-ol-3-one is reduced to the corresponding chlorovinyl derivative (the 21 halo-19.-nor-5(10),20-pregnadiene- 17fl-ol-3-one) on hydrogenation with a Lindlar catalyst, and to the corresponding chlorethyl derivative (the 21- halo-19-nor-5(10)-pregnene-17fi-ol-3-one) on hydrogenation using a heavy metal catalyst such as PtO A further embodiment of our invention comprises novel pharmaceutical compositions containing these 21-haloethisterones and 2l-halonorethisterones.

Various changes and modifications may be made in the present invention, certain preferred embodiments of which are herein disclosed, without departing from the scope thereof; to the extent that these changes and modifications are within the scope of the appended claims, they are to be considered a part of this invention.

Example 1 Twenty mg. of p-toluenesulfonyl chloride is added to 400 mg. of 17u-ethynyl-5-androstene-3fi,17 8-diol in 20 ml. of dihydropyran. The resulting mixture is allowed to stand at room temperature overnight. A 2.5 N NaOH solution is added until the mixture is slightly alkaline. Water is then added and the aqueous phase extracted with 4 portions of ether, each containing approximately 50 ml. The combined ether layers are washed with water, dried over Na SO and evaporated under reduced pressure to give about 725 mg. of a non-crystalline product. The product dissolved in petroleum ether is chromatographed on 60 g. of neutral alumina and the chromatogram eluted with a 7:3 mixture of petroleum ether: ether to give 400 mg. of crystalline product, the 17stethynyl-5-androstene-3fl,17/3-diol bis tetrahydropyranyl ether A solution of about 4 grams of 17ot-ethynyl-5-androstone-3 ,B,17B-diol bis-tetrahydropyranyl ether in 75 ml. of t-butyl alcohol is prepared. About 1.1 equivalents of a 1.0 molar potassium t-butoxide is added and the resulting mixture refluxed for one hour, with stirring, and then cooled. About 1.84 ml. of t-butyl hypochlorite is then added in one portion and the reaction mixture is left stirring at room temperature overnight. About 100 ml. of water is added and the resulting aqueous mixture is extracted with four portions of ether, each containing approximately 200 ml. The combined layers are washed with water, dried over sodium sulfate, filtered and evaporated to dryness in vacuo. The residual material is dissolved in petroleum ether and chromatographed on 120 g. of alumina. Elution with petroleum ether gives about 3.10 grams (a 70% yield) of crystals of 17uc-Chl0- roethynyl-5-androstene-3B,17,6-diol bis-tetrahydropyranyl ether. The crude product shows infrared peaks at 44p and 2.9,u.

A solution of about 3 g. of the 17ot-chloroethynyl-5- androstene-3B,17,B-diol-bis-tetrahydropyranyl ether in 125 ml. of methanol is prepared. To this solution is added 2.5 ml. of concentrated hydrochloric acid and the reaction mixture is stirred for about 1 hour at room temperature. The methanol is then removed by evaporation under reduced pressure until the product crystallizes. Approximately 100 ml. of water is then added and the resulting product is extracted with four portions of ether, each containing about 200 ml. The combined ether extract is washed with water, dried over sodium sulfate and evaporated to a crystalline residue. The residual crystalline material is recrystallized several times from ether to give about 1.58 g. of 17ot-chloroethynyl-S-androstene-3fl,17 8-diol which has the following properties: M.P. 195 C.

Analysis.Calculated for C H O Cl: C, 72.30; H, 8.38; Cl, 10.16. Found: C, 71.64; H, 8.63; Cl, 10.48.

One hundred mg. of 17a-chloroethynyl-5-androstene- 3,d,l7fidiol is dissolved in 1.0 ml. of cyclohexanone and 10 ml. of benzene in a flask fitted with a magnetic stirrer and a reflux condenser. About 5 ml. of the benzene is distilled and a stream of dry nitrogen is passed through the system and maintained throughout the reaction time. Then 0.5 ml. of a 10% solution of aluminum isopropoxide in benzene is added and the reaction mixture is maintained at reflux temperature for 4 hours. The solution is cooled, 5 drops of water are added and the resultant aluminum hydroxide is filtered oil. The filtrate is taken to dryness under reduced pressure. The material is dissolved in ether, filtered, and the filtrate concentrated to give about 37 mg. of crude 17u-chloroethynyl-4-androstene-1'7B-ol-3-one. M.P. 178183 C. Recrystallization from ether gives about 25 mg. of the purified product, M.P. 182-184 C. Chromatography of all mother liquors on 3 g. of alumina and elution of the chromatogram with ether gives an additional 20 mg. of product, M.P. 18l-184 C. Total yield 45 mg. The product has the following properties:

U.V. ALEQ 241mm, 15,000. LR. A231? 2.8, 4.43, 6.0, 6.18

Analysis.-Calculated for CmHzqOgClI C, 72.73; H, 7.85; Cl, 10.22. Found: C, 73.41; H, 7.93; Cl, 10.81.

Example 2 To a solution of 482 mg. of 17a-ethynyl-5-androstene- 3,8,17fl-diol-bis-tetrahydropyranyl ether in 10 ml. of tertiary-butyl alcohol, is added about 1.1 equivalents of a 1.0 molar potassium t-butoxide. The resulting mixture is refluxed for one hour, with stirring, and then cooled. 196 mg. of N-bromosuccinimide is then added and the reaction mixture is stirred at room temperature for about 18 hours. The entire reaction mixture is dissolved in water and then extracted with 3 portions of ether, each containing approximately 50 ml. The combined ether extracts are washed with three portions of a saturated solution of NaHCO each portion containing approximately 25 ml., then with 3 portions of water, each containing about 25 ml. The ether layer is dried over sodium sulfate, filtered and evaporated to dryness. The oily resi due is filtered through 20 g. of aluminum oxide to give 407 mg. of oily material which is dissolved in petroleum ether and chromatographed on 30 g. of acetone activated alkaline alumina. Elution with a 9:1 mixture of petroleum ether and ether yields 87 mg. of 17ubromoethynyl-5- androstene-3fi,l7,G-diol-bis-tetrahydropyranyl ether. An infrared spectrum of this material shows Amax. 4.5a.

To a solution of 17a-bromoethynyl-5-androstene-3fi, 17B-diol-bis-tetrahydropyranyl ether in 40 ml. of methanol is added 0.8 ml. of concentrated HCl, and the reaction mixture is stirred for one hour at room temperature. The methanol is then removed under reduced pressure. Water is added and the resulting solution is extracted with 3 portions of ether, each portion containing approximately 75 ml. The combined ether extracts are Washed three times with approximately 50 ml. of water, dried over sodium sulfate, filtered and evaporated to dryness. The residual material is crystallized to give 230 mg. of 17abromoethynyl-5-androstene-3[3,17,8-diol which has the following properties: M.P. 214215 C.

I.R. m3? 2.7, 2.89, 4.55;;

Analysis.-Calculated for C H O Br: C, 64.10; H, 7.43; Br, 20.32. Found: C, 62.40; H, 7.65; Br, 20.50.

17a-bromoethynyl-5-androstene-3B,17fl-diol mg.) is dissolved in 1.95 ml. of cyclohexanone and 20 ml. of benzene, using a flask fitted with a magnetic stirrer and a reflux condenser. After 3 ml. of benzene is distilled, a stream of dry nitrogen is passed through the system, and maintained throughout the entire reaction time. After cooling to room temperature, there is added 0.98 ml. of a 10% solution of aluminum isopropoxide in benzene,

and the reaction mixture is refluxed for 3 hours and cooled to room temperature. Ten drops of water are added and the reaction mixture is filtered. The filtrate is taken to dryness. The residue is chromatographed on 15 g. of acetone activated acid-washed alumina and 85 mg. of product is eluted with a mixture of seven parts ether to three parts petroleum ether. The Nix-bromoethynyl- 4-androstene-17,8-ol-3-one so obtained has the following properties:

U.V. Riffs 240 III .1, 615,700, LR. Ami? 2.9, 4.51, 6.0, 6.2;;

AnaIysis.Calculated for C H O Br: C, 64.45; H, 6.95; Br, 20.42. Found: C, 65.16; H, 7.30; Br, 21.44.

Example 3 To a solution of one gram of l7a-ethynyl-tl9-nor-4- androstene-17fi-ol-3-one dissolved in 75 ml. of benzene is added 7.5 ml. of ethylene glycol, together with 50 mg. of p-toluenesulfonic acid. The reaction mixture is heated at reflux with a water separator for 20 hours. The reaction mixture is cooled, and about 10 ml. of bicarbonate solution is added. The reaction mixture is extracted with 3 portions of ether, each portion containing about 100 ml. The combined extracts are washed Wit-h Water, dried over sodium sulfate and evaporated to dryness to give a mixture of the A and A -l7a-ethynyl-3-ethylenedioxy 19- nor-androstene-l7fi-ols.

To a solution of the total crude material from the above reaction in 10 ml. of dihydropyran Which has been distilled from sodium metal, is added with stirring, 120 mg. of p-toluenesulfonyl chloride, and the stirring is continued at room temperature for 64 hours, Saturated sodium bi carbonate solution (10 ml.) is then added to the reaction mixture. The mixture is then extracted with 3 portions of ether, each portion containing about 50 ml. The extracts are combined, washed with water and dried over sodium sulfate, and evaporated to dryness in vacuo. The crude oil (about 3.5 g.) is dissolved in petroleum ether and chromatographed on 100 g. of acetone activated alkaline alumina. The ehromatogram is eluted with mixtures of ether and petroleum ether to give 1.043 g. of a mixture of the A and M 17a-ethynyl-3-ethylenedioxy=l9-norandrostene- 17 3-ol-tetra'hydropynanyl ethers. The infrared spectrum shows bands at 2.9a and 9.0,u.

To a stirred solution of 2.130 g. of the mixture of the A and 11 -17a-ethynyl-3-ethylenedioxy-19-nor-androstene-l7;3-ol-tetrahydropyranyl ethers, dissolved in 50 ml. of dry t-butanol is added 515 ml. of 1 M potassium-tbutoxide. The reaction mixture is heated at reflux for ninety minutes and then cooled to room temperature. Then 0.95 ml. of t-butyl hypochlorite is added. The reactlon mixture is left to stir at room temperature for about 16 hours. About 100 ml. of water is added to the reaction mixture and the aqueous solution is extracted with three portions of ether, each portion containing about 200 ml. The extracts are combined, washed with Water, dried over sodium sulfate, filtered and evaporated .to dryness under reduced pressure. About 2.2 g. of crude product is obtained, This is chromatographed on 100 g. of alkaline alumina. The material dissolved in petroleum ether to which a small amount of benzene has been added is chromatographed on alumina and eluted with etherpetroleum ether mixtures. 1.9 g. of a semi-crystalline product, the 17a-chloroethynyl-3-ethylenedioxy-'19-nor- 5[and 5(10)]-androstene-17/8-ol-tetraliydropyranyl ether, is obtained. Infrared absorption s'hows bands at 4.45 4 and strong absorption in the 9-10 region.

To a stirred solution of 1.9 grams of Hot-chloroethynyl- 3-ethylenedioxy 19-nor-5 [and 5 l0) ]-androstene-l7/3-oltetrahydropyranyl dissolved in 41 ml. of methanol is added 3 ml. of concentrated hydrochloric acid and 2. 1 ml. of water. The reaction mixture is stirred at room temperature for 2% hours. The methanol is then removed under reduced pressure. The residue is dissolved in about 200 ml, of ether and washed with water until the washings are neutral. The ether solution is dried over sodium sulfate and evaporated to dryness. The crystalline residue, recrystallized from a mixture of ether and methylene chloride, gives 1.2 g. of Hot-chloroethynyl-19-nor-4-androstene-l7fl-ol-3-one, M. P. 194 200 C. A sample recrystallized for analysis from ethyl acetate has a melting point of 198-201 C. and shows the following properties:

U.V. M32 240 m,., 6 15,000, LR. x3531? 2.9,., 4.48, 6.05, 6.2,.

Analysis. Calcul-ated for C -H O Cl: C, 72.20; H, 7.57; Cl, 10.65. Found: C, 72.27; H, 7.57; Cl, 9.90.

Ten mg. of the mixture of 17a chloroethynyl 3- ethylenedioxy A [and A androstene 17 3 oltetrahydropyranyl ether is treated with 1 cc. of 70% aqueous acetic acid for 1 hour at room temperature. The product is neutralized with aqueous sodium hydroxide and extracted with ether. The ether extract is dried and concentrated in vacuo. The crude product is chromatographed on acid-washed alumina and eluted with mixtures of ether and petroleum ether to give the 17aethynyl-5(10)-androstene-l7flol-3-one.

Example 4 3.2 ml. of potassium t-butoxide is added to 849 mg. of 17a ethynyl 3 ethylenedioxy-l9-nor-5[and 5(10)]- androstene 17B ol tetrahydropryanyl ether, prepared as in Example 3, in 28 ml. of t-butyl-alcohol. This mixture is refluxed for 75 minutes and then cooled to room temperature. 1.136 g. of N-bromosuccinimide is added and the reaction mixture is then stir-red at room temperature for about 17 hours. The reaction mixture is added to about 60 ml. of water and extracted with 3 portions of ether, each portion containing about 75 ml. The ether extracts are combined, washed with 3, 50 ml. portions of saturated sodium bisulfite solution, and then with water until the washings are neutral. The ether solution is dried over sodium sulfate, filtered and evaporated to dryness to yield the crude Not-bromoethynyl- 3-ethylenedioxy 19 nor 5 [and 5 (10)] androstene- 17B ol tetrahydropyranyl ether which may be used in the next step without purification.

The crude steroid is dissolved in 25 ml. of methanol and 0.2 ml. of concentrated hydrochloric acid. The mixture is stirred at room temperature for 2% hours. The methanol is then removed under reduced pressure, water is added, and the reaction mixture is extracted with 3, 75 ml. portions of ether. The ether layer is washed with water, dried over sodium sulfate, filtered and evaporated to dryness. This gives 737 mg. of crude oil which is chromatographed on 44 g. of acetone activated acidwashed alumina. Elution with a mixture of ether and petroleum ether gives a crystalline material which is recrystallized from ether to yield about 75 mg., M.P. 180182 of 170: bromoethynyl 19 nor-4-androstene- 17,8-01-3-one which has the following properties:

U.V. RES 239 m E mol, 16,300, LR. x2333 2854.55 6.1 and 6.2;].

Analysis.Calculated for C H O Br: C, 63.67; H, 6.68. Found: C, 64.11;H, 7.05.

Ten mg. of the mixture of 17a bromoethynyl 3- ethylenedioxy A [and A androstene 17d oltetrahydropyranyl ether is treated with 1 cc. of 70% aqueous acetic acid or 1 hour at room temperature. The product is neutralized with aqueous sodium hydroxide and extracted with ether. The ether extract is dried and concentrated in vacuo. The crude product is chromatographed on acid-washed alumina and eluted with mixtures of ether and petroleum ether to give the 170:- ethynyl-S 10) -androstene-17fi-ol-3-one.

ExampleS Ten grams of 17a chloroethynyl 5 androstene- 3fi,17fl-diol in ml. of tetrahydrofuran is treated with 100 ml. of 0.9 m. monoperphthalic acid in ethyl acetate and allowed to stand overnight at room temperature. The reaction mixture is diluted with ethyl acetate and washed sequentially with aqueous sodium bicarbonate, sodium bisulfite, and sodium bicarbonate solution. The organic layer is dried and concentrated in vacuo. The product is fractionally crystallized from aqueous ethanol to yield the 170a chloroethynyl androstane 35,175- diol-5,6u-oxi-de.

Five grams of 17a-chloroethynyl-androstane-3l3,17B- diol-5,6u-oxide in benzene is added to a reagent prepared from 3.4 g. of magnesium, 10 ml. of methyl iodide and 4-5 ml. of ether. The mixture is stirred and 70 ml. of solvent is removed by distillation. After refluxing for 5 hours, the mixture is cooled, acidified with dilute hydrochloric acid and the organic layer washed to neutrality. The organic phase is dried and concentrated in vacuo. Crystallization from aqueous ethanol gives the 6,8-methyl- 17a-chloroethynyl-androstane-313,5ot,17,3-triol.

The oxidizing reagent is prepared by diluting 2.7 g. of CrO and 2.3 ml. of concentrated sulfuric acid to 10 ml. with water. A solution of 1.90 g. of 6fl-rnethyl-l7otchlorethynyl-androstane-3,8,5ot,17B-triol in 300 ml. of acetone is cooled to and treated with 1.85 ml. of the oxidizing reagent. The solution is diluted with ice water and extracted with ether. The ether extract is washed sequentially with water, and aqueous sodium bicarbonate and then dried and concentrated in vacuo, to given 6,8 methyl 17cc chloroethynyl androstane- 50,l7fl-diOl-3-OI1C. The crude product is dissolved in 150 ml. of methanol, 75 ml. of l N sodium hydroxide is added and the mixture is stirred at room temperature under nitrogen for 24 hours. The mixture is concentrated to half volume under vacuo at 25 C. and then poured into ice water. The product is extracted with ether. The ether layer is washed with water, dried and concentrated. The concentrate is chromatographed on acid-washed alumina and the product eluted with etherpetroleum ether mixtures to yield 6otmethyl-17a-chloroethynyl-4-androstene-17,8-01-3-one.

In accordance with the above procedures, but starting with the 17ot-bromoethynyl-5-androstene 3fi,17,8-dio1 in place of the 60c methyl-17a-chloroethynyl-4-androstenel7B-ol-3-one, there is obtained the 6et-methyl-17ot-bromoethyny1-4-androstene-17/3-ol-3-one.

Example 6 To 100 mg. of 6ot-methyl-17m-chloroethynyl-4-androstene-17/3-ol-3-one in ml. of t-butanol and 0.1 ml. of acetic acid is added 50 mg. of selenium dioxide. The mixture is refluxed under nitrogen for 18 hours; then 50 mg. of selenium dioxide is added and the mixture is refluxed an additional 24 hours. The product is filtered, and the filtrate evaporated to dryness. The residue is extracted with ethyl acetate and the extract is washed successively with aqueous sodium bicarbonate, ammonium sulfide, dilute ammonia water, water, dilute hydrochloric acid and water, and then dried over magnesium sulfate. The extract is treated with activated charcoal and then concentrated to dryness. Crystallization of the residue from a mixture of acetone and ether gives 6a-methyl-17a-chloroethynyl-1,4-androstadiene-17,8-ol-3-one.

In accordance with the above procedures, but starting with the 6a-methyl-17a-bromoethynyl-4-androstene-175- ol-3-one in place of the Got-methyl-17u-chloroethynyl-4- androstene-17B-ol-3-one, there is obtained the 6a-methyl- 17a-bromoethynyl-1,4-androstadiene-17,8-ol-3-one.

Septomyxa afiinis (ATCC 6,737) is inoculated from a slant into 250 ml. shake flasks containing 50 ml. of the following medium: 2% Edamin (Sheflield Farms), 5% glucose, and 0.5% corn steep liquor. After a 48 hour incubation at 28 C., mg. of 60c-Ill6thYl-17a-Chl0l'0- ethynyl-4-androstene-17B-ol-3-one is added to each shake flask as a dimethylformamide solution (100 mg./ml.). After a conversion period of 24 hours, the cells are re- 14 moved by filtration, followed by three successive extractions with equal volume of ethyl acetate. The extracts are combined and concentrated in vacuo.

The 604 methyl 17a-chloroethynyl-1,4-androstadiene- 17B-ol-3-one is readily crystallized from the concentrate. Descending paper chromatography of the product in a system using formamide as the stationary phase and chloroform as the mobile phase, shows some of the steroid substrate, but is largely the somewhat more polar A-dehydrogenation product.

Bacillus sphaericus (ATCC 12,488) is inoculated from a slant into 250 ml. shake flasks containing 50 ml. of the following medium: 2% Edamin (Sheffield Farms), 5% glucose, and 0.5% corn steep liquor. After a 24 hour incubation at 28 C., 10 mg. of 6u-methyl-17a-chloro ethynyl-4-androstene-17fl-ol-3-one is added to each shake flask as a dimethylformamide solution mg./ml.). After a conversion period of 24 hours, the cells are removed by filtration, followed by three successive extractions with equal volume of ethyl acetate. The extracts are combined and concentrated in vacuo.

The 60: methyl 17a-chloroethynyl-1,4-androstadiene- 17fl-ol-3-one, is readily crystallized from the concentrate. Descending paper chromatography of the product in a system using formamide as the stationary phase and chloroform as the mobile phase, shows some of. the steroid substrate, but is largely the somewhat more polar A-dehydrogenation product.

In accordance with the above procedures, but using the 6u-methyl-17a-bromoethynyl-4-androstene-17,8-01-3-one in place of the 6a-methyl-17a-chloroethynyl-4-androstene 17,8-o1-3-one, there is obtained the 6a-methyl-17a-bromoethynyl-1,4-androstadiene-17,8-01-3-one.

Example 7 A suspension of 1 g. of 6a-methyl-17-chloroethynyl-4- androstene-17B-ol-3-one and 2.4 g. of chloranil in 30 ml. of ethyl acetate and 6 ml. of acetic acid is stirred and refluxed for 16 hours. The reaction mixture is cooled and filtered. The product is washed sequentially with aqueous bisulfite, aqueous potassium hydroxide and water. The organic layer is dried and concentrated in vacuo. Chromatography yields 6-methyl-17a-chloroethynyl-4,6-androstadiene-17fl-ol-3-one.

Example 8 To 100 mg. of 6-methyl-17a-chloroethynyl-4,6-androstadiene-17B-ol-3-one in 5 ml. of t-butanol and 0.1 ml. of acetic acid is added 50 mg. of selenium dioxide. The mixture is refluxed under nitrogen for 18 hours; then 50 mg; of selenium dioxide is added and the mixture is refluxed for an additional 24 hours. The product is filtered and the filtrate evaporated to dryness. The residue is extracted with ethyl acetate and the extract is washed successively with aqueous sodium bicarbonate, ammonium sulfide, dilute ammonia water, water, dilute hydrochloric acid and water, and is then dried over magnesium sulfate. The extract is treated with activated charcoal and then concentrated to dryness. Crystallization of the residue from a mixture of acetone and ether gives 6-methyl-17otchloroethynyl-1,4,6-androstatriene-17B-ol-3-one.

In accordance with the above procedures, but starting with the 6-methyl-17a-bromoethynyl-4,6-androstadiene- 17,8-ol-3-one in place of the 6-methyl-17ot-chloroethynyl- 4,6-androstadiene-l7l3-ol-3-one there is obtained the 6- methyl 17cc bromoethynyl-1,4,6-androstatriene--01- 3-one.

Septomyxa afiinis (ATCC 6,737) is inoculated from a slant into 250 ml. shake flasks containing 50 ml. of the following medium: 2% Edamin (Sheflield Farms), 5% glucose, and 0.5% corn steep liquor. After a 48 hour incubation at 28 C., 10 mg. of 6-methyl-l7ot-chloroethynyl-4,6androstadiene-1713-01-3-one is added to each shake flask as a dimethylformamide solution (100 mg./ ml.). After a conversion period of 24 hours, the cells are removed by filtration, followed by three successive extractions with equal volume of ethyl acetate. The extracts are combined and concentrated in vacuo.

The 6 methyl-17a-chloroethynyl-1,4,6-androstatriene- 17B-ol-3-one, is readily crystallized from the concentrate. Descending paper chromatography of the product in a system using formamide as the stationary phase and chloroform as the mobile phase, shows some of the steroid substrate, but is largely the somewhat more polar A- dehydrogenation product.

Bacillus sphaericus (ATCC 12,488) is inoculated from a slant into 250 ml. shake flasks containing 50 ml. of the following medium: 2% Edamin (Sheffield Farms), 5% glucose, and 0.5% corn steep liquor. After a 24 hour incubation at 28 C., mg. of 6oc-I116thYl-l7oc-Chl0l0- ethynyl-4,6-androstadiene-l7fi-ol-3-one is added to each shake flask as a dimethylformamide solution (100 mg./ ml.). After a conversion period of 24 hours, the cells are removed by filtration, followed by three successive extractions with equal volume of ethyl acetate. The extracts are combined and concentrated in vacuo.

The 6a-methyl-l7a-chloroethynyl-1,4,6-androstatriene- 17B-ol-3-one, is readily crystallized from the concentrate. Descending paper chromatography of the product in a system using formamide as the stationary phase, and chloroform as the mobile phase, shows some of the steroid substrate, but is largely the somewhat more polar A-dehydrogenation product.

In accordance with the above procedures, but starting with the 6a-methyl17a-bromoethynyl-4,6-androstadienel7fl-ol-3-one in place of the 6a-methyl-l7a-chloroethynyl- 4,6-androstadiene-17,B-ol-3-one, there is obtained the 6ozmethyl 17a-bromoethynyl-1,4,6-androstatriene-17,8-ol-3- one.

Example 9 A mixture of 500 mg. of the 6a-methy1-17a-chloroethynyl-4-androstene-l7fi-ol-3-one, 10 ml. of dimethyl formamide, 20 ml. of methyl iodide, and 1.5 gms. of silver oxide is stirred at room temperature for 4 days. An additional /2 gm. of silver oxide is added at the end of each day. One hundred ml. of chloroform is then added to the reaction mixture, then stirred for one hour, filtered, and the filtrate evaporated to dryness. The residual oil is chromatographed with acid-washed alumina and eluted with mixtures of ether and petroleum ether to give Got-methyl-17a-chloroethynyl-4-androstene-17pmethoxy-3-one.

In accordance with the above procedures, but starting with the 6u-methyl-l7a-bromoethynyl-4-androstene-17/3- ol-3-one in place of the 6a-methyl-17a-chloroethynyl-4- androstenc-17fl-ol-3-one there is obtained the 6a-methyl 17a-bromoethynyl-4-androstene-17,8-methoxy-3-one.

100 mg. of 6a-methyl-17a-chloroethynyl-4-androstene- 17fi-ol-3-one is heated with 1 cc. of acetic anhydride and 1.2 cc. of pyridine on the steam bath over night. The reaction mixture is then poured onto ice and extracted with chloroform. The extract is washed with water and concentrated. The concentrate is chromatographed over acid-washed alumina and eluted with mixtures of ether and petroleum ether to give 6a-methyl-l7a-chloroethynyl- 4-androstene-17fi-ol-3-one acetate.

In accordance with the above procedures, but starting with the 6u-methyl-l7a-bromoethynyl-4-androstene-17,8- ol-3-one in place of the 6a-methyl-17u-chloroethyny1-4- androstene-17B-ol-3-one there is obtained the 6a-methyl- 17a-bromoethynyl-4-androstene-17 3-ol-3-one acetate.

Example 10 100 mg. of 17a-chloroethynyl-4-androstene-1713-01-3- one (Example 1) is heated with 1 cc. of acetic anhydride and 1.2 cc. of pyridine on the steam bath overnight. The reaction mixture is then poured onto ice and extracted with chloroform. The extract is washed with water and concentrated. The concentrate is chromatographed over acid-washed alumina and eluted with mixtures of ether- 16 petroleum and ether to give 17a-chloroethynyl-4-androstene-17B-ol-3-one acetate.

In accordance with the above procedure, but starting with the 17a-bromoethynyl-4-androstene-176-01-3-one in place of the 17a-chloroethynyl-4-androstene-17B-ol-3-one there is obtained the 17a-bromoethynyl-4-androstene-17/3- ol-3-one acetate.

Example 11 To mg. of 17a-chloroethynyl-4-androstene-175-01- 3-one acetate in 5 ml. of t-butanol and 0.1 ml. of acetic acid is added 50 mg. of selenium dioxide. The mixture is refluxed under nitrogen for 18 hours; then 50 mg. of selenium dioxide is added and the mixture is refluxed an additional 24 hours. The product is filtered, and the fil trate evaporated to dryness. The residue is extracted with ethyl acetate and the extract is washed successively with aqueous sodium bicarbonate, water, dilute hydrochloric acid and water, and then dried over magnesium sulfate. The extract is treated with activated charcoal and then concentrated to dryness. Crystallization of the residue gives 17a chloroethynyl-1,4 androstadiene-l7/3-ol-3-one acetate.

In accordance with the above procedures, but starting with the bromoethynyl-4-androstene 17B-ol-3-one acetate in place of the 17a-chloroethynyl-4-androstene- 17B-ol-3-one acetate, there is obtained the l7a-bromoethynyl-l,4-androstadiene-17fi-ol-3-one acetate Corynebacterium simplex (ATCC 6,946) is inoculated from a slant to 250 ml. shake flasks containing a medium having the composition: 1 g./liter yeast extract (Difco). After an 18 hour growth phase at 28 C., 10 mg. of 17achloroethynyl-4-androstene-17/3-ol-3-one acetate is added to each flask as a dimethylformamide solution (100 mg./ ml.). After a 24 hour transformation period at 28 C., the cells are centrifuged, followed by three ethyl acetate extracts of the cell-free broth. The extracts are combined and concentrated in vacuo. The 17a-chloroethynyl-l,4- androstadiene 17fi-ol-3-one acetate is crystallized directly from the concentrate. Paper chromatography of the product in a system utilizing formamide as the stationary phase and chloroform-benzene (1:1) as the mobile phase indicates that the product possesses a polarity slightly greater than the substrate.

Septomyxa affinis (ATCC 6,737) is inoculated from a slant into 250 ml. shake flasks containing 50 m1. of the following medium: 2% Edamin (Shefifield Farms), 5% glucose, 0.5% corn steep liquor. After a 48 hour incubation at 28 C., 10 mg. of 17u-chloroethynyl-4-androstene-17B-ol-3-one acetate is added to each shake flask as a dimethylformamide solution (100 mg./ml.). After a conversion period of 24 hours, the cells are removed by filtration, followed by three successive extractions with equal volume of ethyl acetate. The extracts are combined and concentrated in vacuo.

The product is re-acetylated by treatment with acetic anhydride and pyridine, and then purified by recrystallization to give the 17a-chloroethyny1-1,4-androstadiene-17,8- ol-3-one acetate. Descending paper chromatography of the product in a system using formamide as the stationary phase, and chloroform as the mobile phase, shows some of the steroid substrate, but is largely the somewhat more polar A-dehydrogenation product.

In accordance with the above procedures, but using the 17u-bromoethynyl-4-androstene -01-3 one acetate in place of the 17a-chloroethynyl-4-androstene-1743-01-3-one acetate, there is obtained the 17a-bromoethynyl-1,4-androstadiene-17fl-ol-3-one acetate.

Example 12 A mixture of 500 mg. of the 17a-chloroethynyl-4-androstene-l7 3-ol-3-one, 10 ml. of dimethylformamide, 20 ml. of methyl iodide, and 1.5 gms. of silver oxide is stirred at room temperature for 4 days. An additional /z gm. Of S l er oxide is added at the end of each day. One hun- 17 dred ml. of chloroform is then added to the reaction mixture, which is then stirred for one hour, filtered, and the filtrate is evaporated to dryness. The residual oil is chromatographed with acid-washed alumina and eluted with mixtures of ether and petroleum ether to give 17achloroethynyl-4-androstene-l7fl-methoxy-3-one.

In accordance with the above procedures, but starting with the 17a-bromoethynyl-4-androstene-17fl-ol-3-one in place of the 17a-chloroethynyl-4-androstene-17fl-ol-3-one, there is obtained the 17a-bromoethynyl-4-androstene-17,8- methoxy-3-one.

Example 13 To 100 mg. of 17a-chloroethynyl-4-androstene-17,8- methoxy-3-one in 5 ml. of t-butanol and 0.1 ml. of acetic acid is added 50 mg. of selenium dioxide. The mixture is refluxed under nitrogen for 18 hours; then 50 mg. of selenium dioxide is added and the mixture is refluxed an additional 24 hours. The product is filtered, and the filtrate evaporated to dryness. The residue is extracted with ethyl acetate and the extract is washed successively with aqueous sodium bicarbonate, water, dilute hydrochloric acid and water, and then dried over magnesium sulfate. The extract is treated with activated charcoal and then concentrated to dryness. Crystallization of the residue from a mixture of acetone and ether gives 17achloroethynyl-1,4-androstadiene17fi-methoxy-3-one.

Septomyxa afiinis (ATCC 6,737) is inoculated from a slant into 250 ml. shake flasks containing 50 ml. of the following medium: 2% Edamin (Shefiield Farms), 5% glucose, and 0.5% corn steep liquor. After a 48 hour incubation at 28 C., mg. of 17ot-chloroethynyl-4- and-rostene-17 8-methoxy-3-one is added to each shake flask as a dimethylformamide solution (100 mg./ml.). After a conversion period of 24 hours, the cells are removed by filtration, followed by three successive extractions with equal volume of ethyl acetate. The extracts are combined and concentrated in vacuo.

The 1704 chloroethynyl 1,4 androstadiene 1713- methoxy-3-one is readily crystallized from the concentrate. Descending paper chromatography of the product in a system using formamide as the stationary phase and chloroform as the mobile phase, shows some of the steroid substrate, but is largely the somewhat more polar A'-dehydrogenation product.

Bacillus sphaericus (ATCC 12,488) is inoculated from a slant into 250 ml. shake flasks containing 50 m1. of the following medium: 2% Edamin (Sheffield Farms), 5% glucose, and 0.5% corn steep liquor. After a 24 hour incubation at 28 C., 10 mg. of 17u-chloroethynyl-4- androstene-17,8-methoxy-3-one is added to each shake flask as a dimethylformamide solution (100 mg./ml.). After a conversion period of 24 hours, the cells are removed by filtration, followed by three successive extractions with equal volume of ethyl acetate. The extracts are combined and concentrated in vacuo.

The 17a chloroethynyl 1,4 androstadiene 17B- rnethoxy-3-one, is readily crystallized from the concentrate. Descending paper chromatography of the product in a system using formamide as the stationary phase and chloroform as the mobile phase, shows some of the steroid substrate, but is largely the somewhat more polar A-dehydrogenation product.

In accordance with the above procedures, but using the 170: bromoethynyl 4 androstene 17B methoxy 3- one in place of the 17ot-chloroethynyl-4-androstene-175- methoxy-3-one, there is obtained the Hot-bromoethynyl- 1,4-androstadiene-17fi-rnethoxy-3-one.

Example 14 A mixture of 1 g. of 17ot-chloroethynyl-4-androstene- 17,8-ol-3-one, 10 ml. of acetic anhydride and 100 mg. of p-toluenesulfonic acid is heated on the steam bath for one hour and allowed to stand overnight at room temperature. It is then poured into ice water and left to 18 stand at room temperature for one half hour. The reaction mixture is then extracted with ether. The ether extract is washed with water and aqueous sodium bicarbonate, dried, and concentrated to yield the crude 170tchloroethynyl 3,5 androstadiene-3,17B-diol diacetate. The product, crystallized from petroleum ether, has a melting point of 125-131 C. The analysis of a sample which has a melting point of 130136 C. is: C, 69.75; H, 7.23. Calculated C, 69.67; H, 7.25.

To a solution of 200 mg. of the Not-chloroethynyl- 3,5-androstadiene-3,17B-diol-diacetate dissolved in 2 ml. of acetic acid is added with stirring, 56 ml. of N-chlorosuccinimide and 2 ml. of tetrahydrofuran containing 5% of dry HCl. The reaction is stirred at room temperature for 2 /2 hours. Sodium bicarbonate is added and the reaction mixture is extracted with ether. The ether extracts are dried and concentrated. The material is chromatographed over acid-washed alumina, and eluted with ether-petroleum ether mixtures, to give the 6mchloro 17a. chloroethynyl-4-androstene-17B-ol-3-one acetate. The analytical sample is crystallized from a mixture of methylene chloride-ether and has the following properties: M.P. 220230 C.

w 235 my, 6 13,600

To 100 mg. of Got-chloro-17ot-chloroethynyl-4-androstene-17B-ol-3-one acetate in 5 ml. of t-butanol and 0.1 ml. of acetic acid is added mg. of selenium dioxide. The mixture is refluxed under nitrogen for 18 hours; then 50 mg. of selenium dioxide is added and the mixture is refluxed an additional 24 hours. The product is filtered, and the filtrate evaporated to dryness. The residue is extracted with ethyl acetate and the extract is washed successively with aqueous sodium bicarbonate, ammonium sulfide, dilute ammonia water, water, dilute hydrochloric acid and water, and then dried over magnesium sulfate. The extract is treated with activated charcoal and then concentrated to dryness. Crystallization of the residue from a mixture of acetone and ether gives 6a-chloro-17otchloroethynyl-1,4-androstadiene-17,8-01-3-one acetate.

In accordance with the above procedures, but starting with the 6a-chloro-17a-bromoethynyl-4-androstene-17B- ol-3-one acetate in place of the 6a-chloro-17a-chloroethynyl-4-androstene-17fl-ol-3-one acetate there is obtained the 6a-chloro-Not-bromoethynyl-1,4-androstadiene- 17fi-ol-3-one acetate.

Corynebacterium simplex (ATCC 6,946) is inoculated from a slant to 250 ml. shake flasks containing a medium having the composition: 1 g./liter yeast extract (Difco). After an 18 hour growth phase at 28 C., 10 mg. of 6achloro 17oz chloroethynyl 4 androstene 17B ol 3-one acetate is added to each flask as a dirnethylformamide solution mg./ml.). After a 24 hour transformation period at 28 C., the cells are centrifuged, followed by three ethyl acetate extracts of the cell-free broth. The extracts are combined and concentrated in vacuo. The 600 chloro 17a chloroethynyl 1,4 -androstadiene 17B-ol-3-one acetate is crystallized directly from the concentrate. Paper chromatography of the product is a system utilizing formamide as the stationary phase and chloroform-benzene (1:1) as the mobile phase indicates that the product possesses a polarity slightly greater than the substrate.

. Septomyxa afilm's (ATCC 6,737) is inoculated from a slant into 250 ml. shake flasks containing 50 ml. of the following medium: 2% Edamin (Sheflield Farms), glucose, and 0.5 corn steep liquor. After a 48 hour incubation at 28 C., mg. of 6oc-Cl1l0fO-17u-Chl0f0- ethynyl-4-androstene-17/3-ol-3-one acetate is added to each shake flask as a dimethylformamide solution (100 mg./ml.). After a conversion period of 24 hours, the cells are removed by filtration, followed by three successive extractions with equal volume of ethyl acetate. The extracts are combined and concentrated in vacuo.

The product is re-acetylated by treatment with acetic anhydride and pyridine, and then purified by recrystallization to give the 6a-chloro-Not-chloroethynyl-1,4-androstadiene-17fi-ol-3-0ne acetate, is readily crystallized from the concentrate. Descending paper chromatgraphy of the product in a system using formamide as the stationary phase and chloroform as the mobile phase, shows some of the steroid substrate, but is largely the somewhat more polar A-dehydrogenation product.

In accordance with the above procedures, but using the 60: chloro 17a bromoethynyl 4 androstene 17B ol-3-one acetate in place of the 6a-chloro-17a-chloroethynyl-4-androstene-17 8-ol-3-0ne acetate, there is obtained the 60: chloro 17cc bromoethynyl-1,4-androstadiene-l7fi-ol-3-one acetate.

Example 16 A mixture of 1 g. of 17u-chloroethynyl-4-androstenel7 3-methoxy-3-one (Example 10), 10 ml. of acetic anhydride and 100 mg. of p-toluenesulfonic acid is heated on the steam bath for one hour and allowed to stand overnight at room temperature. It is then poured into ice water and left to stand at room temperature for one half hour. The reaction mixture is then extracted with ether. The ether extract is washed with water and aqueous sodium bicarbonate, dried, and concentrated to yield the 17m chloroethynyl 3,5 androstadiene 176 methoxy- 3-ol acetate.

To a solution of 200 mg. of the 17a-chloroethynyl-3,5- androstadiene-17fl-methoxy-3-ol-acetate dissolved in 2 ml. of acetic acid is added, with stirring, 56 ml. of N-chlorosuccinimide and 2 ml. of tetrahydrofuran containing 5% of dry HCl. The reaction is stirred at room temperature for 2 /2 hours. Sodium bicarbonate is added. The reaction mixture is then extracted with ether. The ether extracts are dried and concentrated. The material is chromatographed over acid-washed alumina, and eluted with ether-petroleum ether mixtures to give 6oL-ChlOfO-l7a-Chl0- roethynyl-4-androstene-17/3-methoxy-3-one.

In accordance with the above procedures, but starting with the 17a-bromoethynyl-4-androstene-17B-methoxy-3- one in place of the 17ot-chloroethynyl-4-androstene-17,8- methoxy-3-one, there is obtained as intermediates, the 17oz chloroethynyl 3,5 androstadiene 171i methoxy- 3-ol-acetate, and as product, the 6oc-ChlOIO-l7oc-Chl0r0- ethynyl-4-androstene-17,8-methoxy-3-one.

Example 17 To 100 mg. of 17a-chloroethynyl-4-androstene-17B- methoxy-3-one in 5 ml. of t-butanol and 0.1 ml. of acetic acid is added 50 mg. of selenium dioxide. The mixture is refluxed under nitrogen for 18 hours; then 50 mg. of selenium dioxide is added and the mixture is refluxed an additional 24 hours. The product is filtered, and the filtrate evaporated to dryness. The residue is extracted with ethyl acetate and the extract is washed successively with aqueous sodium bicarbonate, ammonium sulfide, dilute ammonia water, water, dilute hydrochloric acid and water, and then dried over magnesium sulfate. The extract is treated with activated charcoal and then concentrated to dryness. Crystallization of the residue from a mixture of acetone and ether gives 17ot-chloroethynyl-1,4- androstadiene-l7p-mcthoxy-3rone,

Septomyxa afiinis (ATCC 6,737) is inoculated from a slant into 250 ml. shake flasks containing 50 ml. of the following medium: 2% Edamin (Sheflield Farms), 5% glucose, and 0.5% corn steep liquor. After a 48 hour incubation at 28 C., 10 mg. of 17a-chloroethynyl-4- androstene-17/3-methoxy-3-one is added to each shake flask as a dimethylformamide solution mg./ml.). After a conversion period of 24 hours, the cells are removed by filtration, followed by three successive extractions with equal volume of ethyl acetate. The extracts are combined and concentrated in vacuo.

The 17e-chloroethynyl-1,4-androstadiene-l7B-methoxy- 3-one is readily crystallized from the concentrate. Descending paper chromatography of the product in a system using formamide as the stationary phase and chloroform as the mobile phase, shows some of the steroid substrate, but is largely the somewhat more polar A-dehydrogenation product.

Bacillus sphaericus (ATCC 12,488) is inoculated from a slant into 250 ml. shake flasks containing 50 ml. of the following medium: 2% Edamin (Sheflield Farms), 5% glucose, and 0.5% corn steep liquor. After a 24 hour incubation at 28 C., 10 mg. of 17a-chloroethynyl-4- androstene-17,8-methoxy-3-one is added to each shake flask as a dimethylformamide solution (100 mg./ml.). After a conversion period of 24 hours, the cells are removed by filtration, followed by three successive extractions with equal volume of ethyl acetate. The extracts are combined and concentrated in vacuo.

The Nix-chloroethynyl-1,4-androstadiene-17B-methoxy- 3-one is readily crystallized from the concentrate. Descending paper chromatography of the product in a system using formamide as the stationary phase and chloroform as the mobile phase, shows some of the steroid substrate, but is largely the somewhat more polar A'-dehy drogenation product.

In accordance with the above procedures, but using the 17:1 bromoethynyl 4 androstene 17B methoxy 3- one in place of the 17a-chloroethynyl-4-androstene-17;?- methoxy-3-one, there is obtained the Hot-bromoethynyll,4-androstadiene-l7fl-methoxy-3-one.

Example 18 A suspension of 1 g. of 17a-chloroethynyl-4-androstene- 17B-ol-3-one and 2.4 g. of chloranil in 30 ml. of ethyl acetate and 6 ml. of acetic acid is stirred and refluxed for 16 hours. The reaction mixture is cooled and filtered. The product is washed sequentially with aqueous bisulfite, aqueous potassium hydroxide and water. Chromatography yields 17a-chloroethynyl-4,6-androstadiene-1713-01-3- one which on crystallization from ethyl acetate has the following properties: M.P. 209-211 C.: a 159 (0. 0.9 CHCl U.V. x3 52 240 m E% 377 A solution of mg. of the oxide in 10 ml. of 0.4 N hydrogen chloride in chloroform is allowed to stand at room temperature for 5.5 hours. The solution is poured onto ice. Aqueous sodium bicarbonate solution is added and the product is extracted with chloroform. The or- Found 21 ganic layer is washed with water, dried and concentrated. Crystallization from ether affords 84 mg. of 6-chloro-l7- chloroethyny1-4,6androstadiene-17B-0l-3-one, M.P. 195- 205 C. The sample for analysis is crystallized from methanol and has the following properties: M.P. 203- 208 C.; a 96 (c. 1.0 CHCl U.V. x9352 285 III/.4, e 21,400

Calculated for C H O Cl C, 66.49; H, 6.38. Found: C, 66.28; H, 6.62.

In accordance with the above procedures, but starting with the 17a-bromoethynyl-4-androstene-17,B-ol-3-one in place of the 17a-chloroethynyl-4-androstene-175-ol-3-one, there is obtained as intermediate, the 17x-bromoethynyl- 4-androstene-17fl-ol-3-one-6,7-oxide, and as product, the 6 chloro 17 br'omoethynyl 4,6 androstadiene 17/3- ol-3-one.

A solution of 125 mg. of 6-chloro-17a-chloroethynyl- 4,6-androstadiene-17}8-ol-3-one in 1.8 ml. of pyridine and 1.5 ml. of acetic anhydride is heated on the steam bath overnight. The solution is poured into ice water and extracted with ether. The ether solution is washed sequentially with dilute hydrochloric acid, water and sodium bicarbonate solution. Chromatography on 7.0 g. of acid-washed alumina, and elution with ether; petroleum ether mixtures affords 56 mg. of 6-chloro-l7a-chloroethynyl-4,6-androstadiene-175-01-3-one acetate. The sample for analysis is crystallized from ether and has the following properties: M.P. 203208 C., (1 98 (c. 0.9 CHC1 U.V. AXQZB 284 my, 6 21,300 Calculated for C H O C1 C, 65.57; H, 6.21. Found: C, 66.06; H, 6.16.

In accordance with the above procedures, but starting with the 6-chloro-17ot-bromoethynyl-4,6-androstadiene- 17fl-ol-3-one in place of the 6-chloro-17ot-chloroethynyl- 4,6-androstadiene-17,8-ol-3-one, there is obtained the 6-chloro 17oz bromoethynyl 4,6 androstadiene 17/3- ol-3-one acetate.

Example 19 To 100 mg. of 6 t-chloro-17a-chloroethynyl-4,6-androstadiene-17B-ol-3-one acetate in ml. of t-butanol and 0.1 ml. of acetic acid is added 50 mg. of selenium dioxide. The mixture is refluxed under nitrogen for 18 hours; then 50 mg. of selenium dioxide is added and the mixture is refluxed an additional 24 hours. The product is filtered, and the filtrate evaporated to dryness. The residue is extracted with ethyl acetate and the extract is washed successively with aqueous sodium bicarbonate, water, dilute hydrochloric acid and water, and then dried over magnesium sulfate. The extract is treated with activated charcoal and then concentrated to dryness. Crystallization of the residue gives 6-chloro-17a-chloroethynyl-1,4,6- androstatriene-17,6-ol-3-one acetate.

Corynebacterium simplex (ATCC 6,946) is inoculated from a slant to 250 ml. shake flasks containing a medium having the composition: 1 g./liter yeast extract (Difco). After an 18 hour growth phase at 28 C., mg. of 6- chloro-l7a-chloroethynyl 4,6 androstadiene-17B-ol-3- one acetate is added to each flask as a dimethylformamide solution (100 mg./ml.). After a 24 hour transformation period at 28 C., the cells are centrifuged, followed by three ethyl acetate extracts of the cell-free broth. The extracts are combined and concentrated in vacuo. The .6-chloro-17ot-chloroethynyl 1,4,6 andostatriene-17B-ol- 3-one acetate is crystallized directly from the concentrate. Paper chromatography of the product is a system utilizing formamide as the stationary phase and chloroformbenzene (1:1) as the mobile phase indicates that the product possesses a polarity slightly greaterthan the substrate.

Septomyxa afiinis (ATCC 6,737) is inoculated from a slant into 250 ml. shake flasks containing 50 ml. of the following medium: 2% Edamin (Sheffield Farms), 5%

glucose, and 0.5% corn steep liquor. After a 48 hour incubation at 28 C., 10 mg. of 6-chloro-17a-chloroethynyl-4,6-androstadiene-17fi-ol-3-one acetate is added to each shake flask as a dimethylformamide solution mg./rnl.). After a conversion period of 24 hours, the cells are removed by filtration, followed by three successive extractions with equal volume of ethyl acetate. The extracts are combined and concentrated in vacuo.

The product is re-acetylated by treatment with acetic anhydride and pyridine, and then purified by recrystallization to give the 6-chloro-l7a-chloroethyny1-1,4,6-androstatriene-17fi-ol-3-one acetate. Descending paper chromatography of the product in a system using formamide as the stationary phase and chloroform as the mobile phase, shows some of the steroid substrate, but is largely the somewhat more polar A-dehydrogenation product.

In accordance with the above procedures, but using the 6-chloro-l7u-bromoethynyl 4,6- androstadiene-17B-ol- 3-one in place of the 6-chlorol7a-chloroethynyl-4,6- androstadiene-l7fl-ol-3-one, there is obtained the 6-chloro- 17a bromoethynyl 1,4,6 androstatriene-17fl-ol-3-one acetate.

Example 20 A mixture of 500 mg. of 6-chloro-17ot-chloro-ethynyl- 4,6-androstadiene-17B-ol-3-one, 10 ml. of dimethylformamide, 20 ml. of methyl iodide, and 1.5 gms. of silver oxide is stirred at room temperature for 4 days. An additional /2 gm. of silver oxide is added at the end of each day. 100 ml. of chloroform is then added to the reaction mixture which is then stirred for one hour, filtered. The filtrate is then taken to dryness. The oil is chromatographed with acid-washed alumina and eluted with mixtures of ether and petroleum ether to give the 6- chloro-17ot-chlor0ethynyl methoxy-4,6-androstadien'e-3-one.

In accordance with the above procedures, but starting with the 6-chloro-17a-bromoethynyl-4,6-androstadiene- 17B-ol-3-one in place of the 6-chloro-17a-chloroethynyl- 4,6-androstadiene-l7fi-ol-3-one there is obtained the 6- chloro-17a-bromoethynyl 17,8 methoxy 4,6 androstadiene-3-one.

Example 21 To 100 mg. of 6-chloro-l7a-chloroethynyll7fi-methoxy-4,6-androstadiene-3-one in 5 ml. of t-butanol and 0.1 ml. of acetic acid is added 50 mg. of selenium dioxide. The mixture is refluxed under nitrogen for 18 hours; then 50 mg. of selenium dioxide is added and the mixture is refluxed an additional 24 hours. The product is filtered, and the filtrate evaporated to dryness. The residue is extracted with ethyl acetate and the extract is washed successively with aqueous sodium bicarbonate, ammonium sulfide, dilute ammonia water, water, dilute hydrochloric acid and water, and then dried over magnesium sulfate. The extract is treated with activated charcoal and then concentrated to dryness. Crystallization of the residue from a mixture of acetone and ether gives the 6-ch1oro-17a-ch1oroethynyl 17B methoxy-1,4,6-androstatriene-3-one.

Septomyxa affinis (ATCC 6,737) is inoculated from a slant into 250 ml. shake flasks containing 50 ml. of the following medium: 2% Edamin (Sheflield Farms), 5% glucose, and 0.5 corn steep liquor. After a 48 hour incubation at 28 C., 10 mg. of 6-chloro-17rx-chloroethynyl- 175'-methoxy-4,6-androstadiene-3-one is added to each shake flask as a dimethylformamide solution (100 mg./ml.). After a conversion period of 24 hours, the cells are removed by filtration, followed by three successive extractions with equal volume of ethyl acetate. The extracts are combined and concentrated in vacuo.

The 6-chloro-17a-chloroethynyl 17B methoxy 1,4,6- androstatriene-3-one is readily crystallized from the concentrate. Descending paper chromatography of the product in a system using formamide as the stationary phase and chloroform as the mobile phase, shows some of the steroid substrate, but is largely the somewhat more polar A'-dehydrogenation product.

Bacillus sphaericus (ATCC 12,488) is inoculated from a slant into 250 ml. shake flasks containing 50 ml. of the following medium: 2% Edamin (Sheflield Farms), glucose, and 0.5% corn steep liquor. After a 24 hour incubation at 28 C., mg. of 6-chloro-17u-chloroethynyl-4,6-androstadiene-17B-methoxy-3-one is added to each shake flask as a dimethylformamide solution (100 mg./ml.). After a conversion period of 24 hours, the cells are removed by filtration, followed by three successive extractions with equal volume of ethyl acetate. The extracts are combined and concentrated in vacuo.

The 6-chloro-17a-chloroethynyl-17B methoxy 1,4,6- androstatriene-3-one is readily crystallized from the concentrate. Descending paper chromatography of the product in a system using formamide as the stationary phase and chloroform as the mobile phase, shows some of the steroid substrate, but is largely the somewhat more polar A'-dehydrogenation product.

In accordance with the above procedures, but using the 6-chloro-17a-bromoethynyl-4,6-androstadiene-l7,8 methoxy-3-one in place of the 6-chloro-17a-chloroethyny1-4,6- androstadiene-17 B-methoxy-3-one, there is obtained the 6- chloro-17a-bromoethynyl-1,4,6-androstatriene-17B methoxy-3-one.

Example 22 A solution of the 17a-chl-oroethynyl-3,S-androstadiene- 36,17B-diol diacetate (Example 10) in aqueous tetrahydrofuran is treated with a slow stream of perchloryl fluoride for one hour and then allowed to stand at room temperature for an additional 2 hours. The reaction mixture is poured on ice and extracted with chloroform. The chloroform layer is washed with aqueous bicarbonate solution, dried and concentrated in vacuo. The crude material is dissolved in acetic acid saturated with gaseous hydrogen chloride and allowed to stand for one hour at room temperature. The reaction mixture is poured on ice and extracted with chloroform. The chloroform layer is washed with aqueous bicarbonate solution, dried and concentrated in vacuo. The concentrate is chromat-ographed on acid-washed alumina to yield the 6a-fl11OIO-17ct-Chl010- ethynyl-4-androstene-17/3-ol-3-one acetate.

In accordance with the above procedures, but starting with the 17a-bromoethynyl-3,S-andr-ostadiene-Bfi,17,8-diol diacetate in place of the 17a-chloroethynyl-3,S-androstadiene-3fi,17B-diol diacetate there is obtained the 6a-fluoro- 17a-bromoethynyl-4-androstene-'17fi-ol-3-one acetate.

Example 23 To 100 mg. of 6a-fluoro-17a-chloroethynyl-4-androstene-17B-ol-3-one acetate in 5 ml. of t-butanol and 0.1 ml. of acetic acid is added 50 mg. of selenium dioxide. The mixture is refluxed under nitrogen for 18 hours; then 50 mg. of selenium dioxide is added and the mixture is refluxed an additional 24 hours. The product is filtered, and the filtrate evaporated to dryness. The residue is extracted with ethyl acetate and the extract is washed successively with aqueous sodium bicarbonate, water, dilute hydrochloric acid and water, and then dried over magnesium sulfate. The extract is treated with activated charcoal and then concentrated to dryness. Crystallization of the residue from a mixture of acetone and ether gives 6efluoro-17a-chloroethynyl-1,4-androstadiene-17(3-ol-3 one acetate.

Corynebacterium simplex (ATCC 6,946) is inoculated from a slant to 250 ml. shake flasks containing a medium having the composition: 1 g./liter yeast extract (Difco). After an 18 hour growth phase at 28 C., 10 mg. of 60afluoro-17a-chloroethynyl-4-androstene-17 3-ol-3 one acetate is added to each flask as a dimethylformamide solution (100 mg./ml.). After a 24 hour transformation period at 28 C., the cells are centrifuged, followed by three ethyl acetate extracts of the cell-free .broth. The extracts are combined and concentrated in vacuo. The 60:-

fluoro-17a-chloroethynyl-1,4-androstadiene-17B-ol-3 one acetate is crystallized directly from the concentrate. Paper chromatography of the product is a system utilizing formamide as the stationary and chloroform-benzene (1:1) as the mobile phase indicates that the product possesses a polarity slightly greater than the substrate.

Septomyxa afinl's (ATCC 6,737) is inoculated from a slant into 250 ml. shake flasks containing 50 ml. of the following medium: 2% Edamin (Sheflleld Farms), 5% glucose, and 0.5% corn steep liquor. After a 48 hour incubation at 28 C., 10 mg. of 6oc-fiL10IO-17oc-Chl0f06thynyl-4-androstene-17fi-ol-3-one acetate is added to each shake flask as a dimethylformamide solution mg./ ml). After a conversion period of 24 hours, the cells are removed by filtration, followed by three successive extractions with equal volume of ethyl acetate. The extracts are combined and concentrated in vacuo.

The product is re-acetylated by treatment with acetic anhydride and pyridine, and then purified by recrystallization to give the 6ot-fluoro-17a-chloroethynyl-1,4-androstadiene-17/3-ol-3-one acetate. Descending paper chromatography of the product in a system using formamide as the stationary phase and chloroform as the mobile phase, shows some of the steroid substrate, but is largely the somewhat more polar A'-dehydrogenation product.

In accordance with the above procedures, but using the 60 fiuoro 17a-bromoethynyl-4-androstene-17B-ol-3-one acetate in place of the 6u-fluoro-17a-chloroethynyl-4-androstene-l7p-ol-3-one acetate there is obtained the Gafluoro 17a-bromoethynyl-1,4-androstadiene-17/3-ol-3-one acetate.

Example 24 A suspension of 1 g. of 6a-fluoro-17a-chloroethynyl-4- androstene-17fl-ol-3-one acetate, 2.4 g. chloranil, 30 ml. ethyl acetate and 6 ml. of acetic acid is stirred and refluxed for 16 hours. The reaction mixture is cooled and filtered. The product is washed sequentially with aqueous bisulfite, aqueous potassium hydroxide and water.

To 100 mg. of 6-fluoro-17u-chloroethynyl-4,6-androstadiene-17B-ol-3-one acetate in 5 ml. of t-butanol and 0.1 ml. of acetic acid is added 50 mg. of selenium dioxide. The mixture is refluxed under nitrogen for 18 hours; then 50 mg. of selenium dioxide is added and the mixture is refluxed an additional 24 hours. The product is filtered, and the filtrate evaporated to dryness. The residue is extracted with ethyl acetate and the extract is washed successively with aqueous sodium bicarbonate, water, dilute hydrochloric acid and water, and then dried over magnesium sulfate. The extract is treated with activated charcoal and then concentrated to dryness. Crystallization of the residue gives 6-fluoro-17a-chloroethynyl-l,4,6- androstatriene-17,8-01-3-one acetate.

In accordance with the above procedures, but starting with the 6-fluoro-17a-bromoethynyl-4,6-androstadiene- 17fi-ol-3-one acetate in place of the 6-fluoro-17a-chl0roethynyl-4,6-androstadiene-17,8-ol-3-one acetate, there is obtained the 6 fluoro-17a-bromoethynyl-1,4,6-androstatriene-l7 3-ol-3-one acetate.

Corynebacterium simplex (ATCC 6,946) is inoculated from a slant to 250 ml. shake flasks containing a medium having the composition: 1 g./liter yeast extract (Difco). After an 18 hour growth phase at 28 C., 10 mg. of 6- fluoro 17ot-chl0roethynyl-4,6-androstadiene-17/3-ol-3-one acetate is added to each flask as a dimethylformamide solution (100 mg./ml.). After a 24 hour transformation period at 28 C., the cells are centrifuged, followed by three ethyl acetate extracts of the cell-free broth. The extracts are combined and concentrated in vacuo. The 6-fiuoro-17a-chloroethynyl-1,4,6-androstatriene 17B- ol-3-one acetate is crystallized directly from the concentrate. Paper chromatography of the produce in a system utilizing formamide as the stationary phase and chloroform-benzene (1:1) as the mobile phase indicates that the product possesses a polarity slightly greater than the substrate.

Septomyxa afifnzs (ATCC 6,737) is inoculated from a slant into 250 ml. shake flasks containing 50 ml. of the following medium: 2% Edamin (Sheffield Farms), 5% glucose, and 0.5% corn steep liquor. After a 48 hour incubation at 28 C., mg. of 6'-fiuoro-l7ot-chloroethynyl- 4,6-androstadiene-17fi-ol-3-one acetate is added to each shake flask as a dimethylformamide solution (100 mg./ ml.). After a conversion period of 24 hours, the cells are removed by filtration, followed by three successive extractions with equal volume of ethyl acetate. The extracts are combined and concentrated in vacuo.

The product is re-acetylated by treatment with acetic anhydride and pyridine, and then purified by recrystallization to give the 6a-fluoro-l7a-chloroethynyl-l,4,6-androstatriene 175 ol 3-one acetate. Descending paper chromatography of the product in a system using formamide as the stationary phase and chloroform as the mobile phase, shows some of the steroid substrate, but is largely the somewhat more polar A-dehydrogenation product.

In accordance with the above procedures, but using the G-fluoro-17u-bromoethynyl-4,6-androstadiene-1 7B ol 3- one acetate in place of the 6-fluoro-1-7ot-chloroethynyl-4,6- androstadiene-l7,8-ol-3-one acetate, there is obtained the 6-fluoro-l7 a-bromoethynyH,4,6-androstatriene-17 8-01 3- one acetate.

Example 26 A solution of the 17a-chloroethynyl-3,S-androstadiene- 17,8-methoxy-313-ol in aqueous tetrahydrofuran is treated with a slow stream of perchloryl fluoride for one hour and then allowed to stand at room temperature for an additional 2 hours. The reaction mixture is poured on ice and extracted with chloroform. The chloroform layer is washed with aqueous bicarbonate solution, dried and concentrated in vacuo. The crude material is dissolved in acetic acid saturated with gaseous hydrogen chloride and allowed to stand for one hour at room temperature. The reaction mixture is poured on ice and extracted with chloroform. The chloroform layer is washed with aqueous bicarbonate solution, dried and concentrated in vacuo. The concentrate is chromatographed on acid-washed alumina to yield the 6a-fluoro-l7a-chloroethynyl-4-andro stene-17B-methoxy-3-one.

In accordance with the above procedures, but starting with the 17ot-bromoethynyl-3,S-androstadiene-17fi-methoxy-BB-ol in place of the 17a-chloroethynyl-3,S-androstadiene-l7fl-methoxy- 35-ol, there is obtained the 6a-fluoro 17a-bromoethynyl-4-androstenel7fi-methoXy-3 -one.

Example 27 To 100 mg. of 6a-fluoro-l7a-chloroethynyl-4-androstene-17fi-methoxy-3-one in 5 ml. of t-butanol and 0.1 ml. of acetic acid is added 50 mg. of selenium dioxide. The mixture is refluxed under nitrogen for 18 hours; then 50 mg. of selenium dioxide is added and the mixture is refluxed an additional 24 hours. The product is filtered, and the filtrate evaporated to dryness. The residue is extracted with ethyl acetate and the extract is washed successively with aqueous sodium bicarbonate, water, dilute hydrochloric acid and water, and then dried over magnesium sulfate. The extract is treated with activated charcoal and then concentrated to dryness. Crystallization of the residue gives 6ot-fiuoro-17a-chloroethynyl-1,4-androstadiene-l7B-methoxy-3-one.

Sept'omyxa afiinis (ATCC 6,737) is inoculated from a slant into 250 ml. shake flasks containing 50 ml. of the following medium: 2% Edamin (Sheflield Farms), 5% glucose, and 0.5% corn steep liquor. After a 48 hour incubation at 28 C., '10 mg. of 6a-fluoro-17a-chloroethynyl- 4-androstene-17/3-methoxy-3-one is added to each shake flask as a dimethylformamide solution mg./ml.). After a conversion period of 24 hours, the cells are removed by filtration, followed by three successive extractions with equal volume of ethyl acetate. The extracts are combined and concentrated in vacuo.

The GOL-flHOIO-I70L-ChlOl'Oethynyl-1,4-31'ldIOStfidl6HB-17flmethoxy-3-one is readily crystallized from the concentrate. Descending paper chromatography of the product in a system using formamide as the stationary phase and chloroform as the mobile phase, shows some of the steroid substrate, but is largely the somewhat more polar A'-dehydrogenation product.

Bacillus sphaericus (AT CC 12,488) is inoculated from a slant into 250 m1. shake flasks containing 50 ml. of the following medium: 2% Edamin (Sheffield Farms), 5% glucose, and 0.5% corn steep liquor. After a 24 hour incubation at 28 C., 10 mg. of 6a-flt10l'O-l7ot-Chl0l0- ethynyl-4-androstene-17B-methoxy-3-one is added to each shake flask as a dimethylformamide solution (100 mg./ ml.). After a conversion period of 24 hours, the cells are removed by filtration, followed by three successive extractions with equal volume of ethyl acetate. The extracts are combined and concentrated in vacuo.

The Got-fluoro-l7a-chloroethynyl-1,4-androstadiene-l7fimethoxy-3-one is readily crystallized from the concentrate. Descending paper chromatography of the product in a system using forrnamide as the stationary phase and chloroform as the mobile phase, shows some of the steroid substrate, but is largely the somewhat more polar A'-dehydrogenation product.

In accordance with the above procedures, but using the one in place of the 6a-fluoro-17ot-chloroethynyl-4-androstene-l7fl-methoXy-3-one, there is obtained the 6a-fiuoro- 17a-bromoethynyl-1,4-androstadiene=17Bmethoxy-3-one.

Example 28 A suspension of 1 g. of 6a-fluoro-17a-chloroethynyl-4- androstene-l7fl-methoxy-3-one, 2.4 g. chloranil, 30 ml. ethyl acetate and 6 ml. of acetic acid is stirred and refiuxed for 16 hours. The reaction mixture is cooled and filtered. The product is washed sequentially with aqueous hisulfite, aqueous potassium hydroxide and water. The organic layer is dried and concentrated in vacuo. Chromatography yields 6-fluoro-17u-chloroethynyl-4,6- androstadiene-17/3-methoxy-3-one.

Example 29 To 100 mg. of 6-fiuoro-l7ot-chloroethynyl-4,6-androstadiene-17B-methoxy-3-one in 5 ml. of t-butanol and 0.1 ml. of acetic acid is added 50' mg. of selenium dioxide. The mixture is refluxed under nitrogen for 18 hours; then 50 mg. of selenium dioxide is added and the mixture is refluxed an additional 24 hours. The product is filtered, and the filtrate evaporated to dryness. The residue is extracted with ethyl acetate and the extract is washed successively with aqueous sodium bicarbonate, water, dilute hydrochloric acid and water, and then dried over magnesium sulfate. The extract is treated with activated charcoal and then concentrated to dryness. Crystallization of the residue gives 6-fiuoro-17a-chloroethynyl-1,4,6-androstatriene-17fi-methoxy-3-one.

Septomyxa afifnis (ATCC 6,737) is inoculated from a slant into 250 ml. shake flasks containing 50 ml. of the following medium: 2% Edamin (Shefiield Farms), 5% glucose, and 0.5% corn steep liquor. After a 48 hour incubation at 28 C., 10 mg. of 6-fluoro-17u-chloroethynyl-4,6-androstadiene-17fl-methoxy-3-one is added to each shake flask as a dimethylformamide solution (100 mg./

27 ml.). After a conversion period of 24 hours, the cells are removed by filtration, followed by three successive extractions with equal volume of ethyl acetate. The extracts are combined and concentrated in vacuo.

The 6-fluOI'O-170t-ChlOI'O6thYI1Yl 1,4,6 androstatriene- 17,8-methoxy-3-one is readily crystallized from the concentrate. Descending paper chromatography of the prod net in a system using formamide as the stationary phase and chloroform as the mobile phase, shows some of the steroid substrate, but is largely the somewhat more polar A'-dehydrogenation product.

Bacillus sphaericus (ATCC 12,488) is inoculated from a slant into 250 ml. shake flasks containing 50 ml. of the following medium: 2% Edamin (Sheffield Farms), 5% glucose, and 0.5% corn steep liquor. After a 24 hour incubation at 28 C., mg. of 6-fluoro-17a-chloroethynyl-4,6-androstadiene-17B-methoxy-3-one is added to each shake flask as a dimethylformamide solution (100 mg./ ml.). After a conversion period of 24 hours, the cells are removed by filtration, followed by three successive extractions with equal volume of ethyl acetate. The extracts are combined and concentrated in vacuo.

The 6 fluoro-Not-chloroethynyl-1,4,6-androstatrienel7 3-methoxy-3-one is readily crystallized from the concentrate. Descending paper chromatography of the product in a system using formamide as the stationary phase and chloroform as the mobile phase, shows some of the steroid substrate, but is largely the somewhat more polar A'-dehydrogenation product.

In accordance with the above procedure, but using the 6a-fluoro 17a bromoethynyl 4,6 androstadiene 17B- methoxy-B-one in place of the Ga-fiUOIO-l'lct-ChlOIOClhY- nyl-4,6 androstadiene 17B methoxy-B-one, there is obtained the 6-fluoro-17a-bromoethynyl-1,4,6-androstatriene- 17fl-methoxy-3-one.

Example 30 A solution of 100 mg. of the 17a-chloroethynyl-4-androstene-17fl-ol-3-one and 50 mg. of Lindlar catalyst in 10 cc. of ethyl acetate is treated with hydrogen until one mole of hydrogen has been absorbed. The mixture is filtered and concentrated. Chromatography yields the pure product 21-chloro-4,20-pregnadiene-l75-ol-3-one.

In accordance with the above procedures, but starting with the 17a-bromoethynyl-4-androstene-1713-01-3-one in place of the 17a-chloroethynyl-4-androstene-17fl-ol-3-one there is obtained the 21-chloro-4,20-pregnadiene-1713-01-3- one.

A suspension of platinum oxide in 10 cc. of ethanol is reduced and 100 mg. of 17a-chloroethynyl-4-androstene- 17B-ol-3-one is added. Reduction proceeds until two moles of hydrogen have been absorbed. The solution is filtered, concentrated and chromatographed on alumina to yield the 21-chloro-4-pregnene-17,8-ol-3-one.

In accordance with the above procedures, but starting with the 1704 bromoethynyl 4 androstene 1713 ol- 3-one in place of the 17a-chloroethynyl-4-androstene-l7B- ol-3-one there is obtained the 21-chloro-4-pregnene-17flol-3-one.

Example 31 A mixture of 1 g. of 17a-chloroethynyl-19-n0r-4- androstene-l7B-ol-3-one, 10 ml. of acetic anhydride and 100 mg. of p-toluenesulfonic acid is heated on the steam bath for one hour and allowed to stand overnight at room temperature. It is then poured into ice water and left to stand at room temperature for one half hour. The reaction mixture is then extracted with ether. The ether extract is washed with water and aqueous sodium bicarbonate, dried, and concentrated to yield the crude 17a chloroethynyl 19 nor 3,5 androstadiene 3, 17fi-diol diacetate.

A solution of 8 gms. of 170t-ChlOI'OCthYHY1-19I]O1'3,5 androstadiene-3,17/3-diol diacetate in a mixture of 700 ml. of 95% ethanol and 300 ml. of tetrahydrofuran is cooled to 10 C. and added dropwise, with occasional stirring, over a one hour period, to a cold (0 C.) solution of 18g. sodium borohydride in 400 ml. of ethanol, the reaction temperature not being allowed to exceed 5 C. After completion of the addition, the solution is held at 0-5 C. for an additional two hours. A solution of NaH PO is then added to adjust the pH to about 5. The mixture is then concentrated in vacuo to a small volume, diluted with water and extracted with chloroform. The extract is washed with water, dried and concentrated. The concentrate is chromatographed over acid-washed alumina and eluted with ether-petroleum ether mixtures to give 17a-chloroethynyl-l9-nor-5- androstene-35,17fi-diol 17-acetate.

Ten grams of Hut-chloroethynyl-19-nor-5-androstene- 35,17B-diol-17-acetate in 150 ml. of tetrahydrofuran is treated with ml. of 0.9 m. monoperphthalic acid in ethyl acetate and allowed to stand over night at room temperature. The reaction mixture is diluted with ethyl acetate and washed sequentially with aqueous sodium bicarbonate, sodium bisulfite, and sodium bicarbonate solution. The organic layer is dried and concentrated in vacuo. The product is fractionally crystallized from aqueous ethanol to yield the Not-chloroethynyl-19-norandrostane-3B,17B-diol-5,6a-oxide-17-acetate.

Five grams of 17a-chlor0ethyny1-19-nor-androstane- 3,8,l7B-diol-5,6a-oxide-17-acetate in benzene is added to a reagent prepared from 3.4 g. of magnesium, 10 ml. of methyl iodide and 45 ml. of ether. The mixture is stirred and 70 ml. of solvent is removed by distillation. After refluxing for 5 hours, the mixture is cooled, acidified with dilute hydrochloric acid and the organic layer washed to neutnality. The organic phase is dried and concentrated in vacuo. Crystallization from aqueous ethanol gives the 6B-methyl-Hot-chloroethynyl-19-norandrostane-36,511,17fi-triol.

The oxidizing reagent is prepared by diluting 2.7 g. of C 0 and 2.3 ml. of concentrated sulfuric acid to 10 ml. with water. A solution of 1.90 g. of 6;3-methyl- 17a chloroethynyl 19 nor androstane 6/3,5a,17,8- triol in 300 ml. of acetone is cooled to 0 and treated with 1.85 ml. of the oxidizing reagent. is diluted with ice water and extracted with ether. The ether extract is washed sequentially with water, and aqueous sodium bicarbonate and then dried and concentrated in vacuo, to give 6,8-methyl-17a-chloroethynyll9-nor-androstane-5a,17/3-diol-3-one. The crude product, dissolved in ml. of methanol, 75 ml. of 1 N sodium hydroxide is added and the mixture is stirred at room temperature under nitrogen for 24 hours. The mixture is concentrated to half volume under vacuo at 25 C. and then poured into ice water. The product is extracted with ether. The ether layer is washed with water, dried and concentrated. The concentrate is chromatographed on acid-washed alumina and the product eluted with ether-petroleum ether mixtures to yield 6a-methyl- 317a chloroethynyl 19 nor 4 androstene 1718 01- -one.

In accordance with the above procedures, but starting with the bromoethynyl 19 nor 4 androstene- 176 ol 3 one in place of the 17m chloroethynyl- 19-nor 4-androstene-17/8-ol-3-one there is obtained as intermediates, the 17oz bromoethynyl 19 nor 3,5- androstadiene 3,175 diol diacetate, 17a bromoethynyl 19 nor 5 androstene 3,8,17B diol, 17abromoethynyl 19 nor androstane 35317;? diol 5,60- oxide 17 acetate, and 6/3 methyl 17cc bromoethynyl 19 nor androstane 3,851,175 triol, and as product, the 60: methyl 17a bromoethynyl 19 nor- 4-androstene-17B-ol-3-one.

Example 32 100 mg. of 6a methyl-l7a-chloroethynyl-l9-nor-4- androstene-l7fi-ol-3-one is heated with 1 cc. of acetic anhydride and 1.2 cc. of pyridine on the steam bath overnight. The reaction mixture is then poured onto ice The solution- 29 and extracted With chloroform. The extract is washed with water and concentrated. The concentrate is chromatographed over acid-washed alumina and eluted with mixtures of ether-petroleum and ether to give 60: methyl 17oz chloroethynyl 19 nor 4 androstene-17fi-ol-3-one acetate.

In accordance with the above procedures, but starting with the 6a-methyl-17u-bromoethynyl-19-nor-4-androstene-l7fl-ol-3-0ne in place of the 6a-methyl-17a-chloroethynyl 19 nor 4 androstene 17,8 ol 3 one, there is obtained the 6a-methyl-17ot-bromoethyny1-19- nor-4-androstene-17B-ol-3-one acetate.

A mixture of 500 mg. of the 6a-methyl-17a-chloroethynyl-19-nor-4-androstene-17,8-ol-3-one, 10 ml. of dimethylformamide, 20 ml. of methyl iodide, and 1.5 grns. of silver oxide is stirred at room temperature for 4 days. An additional /2 gm. of silver oxide is added at the end of each day. One hundred ml. of chloroform is then added to the reaction mixture which is then stirred for one hour, filtered, and the filtrate is evaporated to dryness. The residual oil is chromatographed with acidwashed alumina and eluted with mixtures of ether and petroleum ether to give 6a-methyl-17a-chloroethynyl- 19-nor-4-androstene-17fi-meth-oxy-3-one.

In accordance with the above procedures, but starting with the 6ot-methyl-l7a-brom'oethynyl-l9-nor-4-andro stene-17fl-ol-3-one in place of the 6a-methyl-17a-chloroethynyl-19-nor-4-androstene-l7B-ol-3-one, there is obtained the oat-methyl-17ot-bromoethyny1-19-nor-4-androstene-17/3-methoxy-3-one.

Example 33 To a solution of 200 mg. of the 17a-chloroethynyl-19- nor-3,5-androstadiene-3,17,B-diol diacetate (Example 17) dissolved in 2 ml. of acetic acid is added, with stirring, 56 ml. of N-chlorosuccinimide and 2 ml. of tetrahydrofuran containing of dry HCl. The reaction is stirred at room temperature for 2 /2 hours. Sodium bicarbonate is added. The reaction mixture is then extracted with ether. The ether extracts are dried and concentrated. The material is chromatographed over acid-washed alumina, and eluted with ether-petroleum ether mixtures to give 6a-chloro-17at-chloroethynyl-19-nor-androstene-17B-ol-3-one acetate.

In accordance with the above procedures, but starting with the 17ot-bromoethynyl-19-nor-3,S-androstadiene- 3,17fi-diol diacetate in place of the 17a-chloroethynyl-19- nor-3,5-androstadiene-3,17B-diol diacetate there is obtained the 6ix-chloro-l7a-bromoethynyl-l9 nor-androstene-17fl-ol-3-one acetate.

Example 34 A solution of the 17a-chloroethynyl-19-nor-3,5-androstadiene-3,17fi-diol diacetate in aqueous tetrahydrofuran is treated with a slow stream of perchloryl fluoride for one hour and then allowed to stand at room temperature for an additional 2 hours. The reaction mixture is poured on ice .and extracted with chloroform. The chloroform layer is washed with aqueous bicarbonate solution, dried and concentrated in vacuo. The crude material is dissolved in acetic acid saturated with gaseous hydrogen chloride and allowed to stand for one hour at room temperature. The reaction mixture is poured on ice and extracted with chloroform. The chloroform layer is washed with aqueous bicarbonate solution, dried and concentrated in vacuo. The concentrate is chromatographed on acid-washed alumina to yield the 60c fluoro 17oz chloroethynyl 19 nor androstene- 17fl-ol-3-one acetate.

In accordance with the above procedures, but starting with the 17u-bromoethynyl-1'9-nor-3,5-androstadiene- 3,17,8-diol diacetate in place of the 17ot-chloroethyny1-19- nor-3,5-androstadiene-3,17B diol diacetate there is obtained the 60c fluoro-lh-brornoethynyl-19-nor-4-androstene-17fl-ol-3-one acetate.

A mixture of 500 mg. of the 17a-chloroethynyl-19-nor-4- androstene-17,8-ol-3-one (Example 3) 10 ml. of dimethylformamide, 20 ml. of methyl iodide, and 1.5 gm. of silver oxide is stirred at room temperature for 4 days. An additional /2 gm. of silver oxide is added at the end of each day. One hundred ml. of chloroform is then added to the reaction mixture which is then stirred for one hour, filtered, and the filtrate is evaporated to dryness. The residual oil is chromatographed with acid-washed alumina and eluted with mixtures of ether and petroleum ether to give 17a-chloroethynyl-19-nor-4-androstene-17,6-methoxy- 3-one.

In accordance with the above procedures, but starting with the 17otbromoethynyl-19-nor-4-androstene-175-01-3- one in place of the Nix-chloroethynyl?19-nor-4-androstene-17/i-ol-3-one there is obtained the l7a-br-omoethynyl-19-nor-4-androstene-17B-methoxy-3 -one.

stene-17 8-methoxy-3-one, 10 ml. of acetic anhydride and mg. of petoluenesulfonic acid is heated on the steam bath for one hour and allowed to stand overnight at room temperature. It is then poured into ice water and left to stand at room temperature for one half hour. The reaction mixture is then extracted with ether. The ether extract is wa-shed with water and aqueous sodium bicarbonate, dried, and concentrated to yield the crude 17achloroethynyl 19 nor 3,5 androstadiene 17,8-methoxy-3-ol acetate.

-bromoethynyl 19 nor 3,5 androstadiene 17p meth- -oxy-3-ol acetate.

To a solution of 200 mg. of the 17-chloroethynyl-19- nor-3,5-androstadiene-17B-methoxy-3-ol acetate dissolved in 2 ml. of acetic acid is added, with stirring, 56 ml. of N-chlorosuccinimide and 2 ml. of tetrahydrofuran containing 5% of dry HCl. The reaction is stirred at room temperature for 2 /2 hours. Sodium bicarbonate is added. The reaction mixture is then extracted with ether. The ether extracts are dried and concentrated. The material is chromatographed over acid-washed alumina, and eluted with ether-petroleum ether mixtures to give Got-chloro- 17oz chloroethynyl 19 nor 4 androstene 17 8 methoxy-3-one.

In accordance with the above procedures, but starting with the 17a-bromoethynyl-19-nor-3,5-androstadiene-l7fimethoxy-3-ol acetate in place of the Not-chloroethynyl- 19-nor-3,S-androstadiene-17B-methoxy-3-ol acetate there is obtained the 6a-chloro-17a-bromoethynyl-19-nor-4-androstene-17fl-methoxy-3-one.

Example 35 A solution of the Hot-chloroethynyl-19-nor-3,5-androstadiene-17l3-methoxy-3-ol acetate in aqueous tetrahydrofuran is treated with a slow stream of perchloryl fluoride for one hour and then allowed to stand at room temperature for an additional 2 hours. The reaction mixture is poured on ice and extracted with chloroform. The chloroform layer is washed with aqueous bicarbonate solution, dried and concentrated in vacuo. The crude material is dissolved in acetic acid saturated with gaseous hydrogen chloride and allowed to stand for one 'hour at room temperature. The reaction mixture is poured on ice and extracted with chloroform. The chloroform layer is washed with aqueous bicarbonate solution, dried and concentrated in vacuo. The concentrate is chromatographed on acid-washed alumina to yield the 6a-fluoro-'17a-chlor0- ethynyl-l9-nor-4-androstene-17B-methoxy-3-one.

In accordance with the above procedures, but starting with the 17ot bromoethynyl-19-nor-3,5-androstadiene-17fimethoxy-B-ol acetate in place of the 17a-chloroethynyl-19- nor-3,5-androstadiene-17,8-methoxy-3-ol acetate, there is obtained the 6a-fiuoro-17a-bromoethynyl-l9-nor-4-androstene- 17 ,B-methoxy-3-one.

Example 36 -A solution of 100 mg. of the 17a-chloroethynyl-l9-nor- 4-androstene-17B-ol-3one and 50 mg. of Lindlar catalyst in 10 cc. of ethyl acetate is treated with hydrogen until one mole of hydrogen has been absorbed. The mixture is filtered and concentrated to yield the crude chlorovinyl compound. Chromatography yields the pure product 21- chloro-19-n0r-4,20-pregnadiene-l7,B-ol-3-one.

In accordance with the above procedure, but starting with the l7a-bromoethyny1-19-nor-4-androstene-1718-01-3- one, the 17a-chloroethynyl-19-nor-5(10)-androstene-l7fiol-3-one, or the 17u-bromoethynyl-19-n0r-5(l0)-androstene-17B-ol-3-one, in place of the 17a-chloroethynyl-19- nor-4-androstene-l7fi-ol-3-one, there is obtained the 21- bromo 19-nor-4,20-pregnadiene-175-o1-3-one, 21-chloro- 19-nor-5(10),20-pregnadiene-17 8-ol-3-one, or 21-bromo- 19-nor-5 10) ,20-pregnadiene-17 8-ol-3-one respectively.

Example 37 A suspension of platinum oxide in 10 cc. of ethanol is reduced and 100 mg. of 17a-chloroethynyl-19-n0r-4-androstene-17/3-ol-3-one is added. Reduction proceeds until two moles of hydrogen have been absorbed. The solution is filtered, concentrated and chromatographed on alumina to yield the 21-chloro-19-nor-4-pregnene-17fiol-3-one.

In accordance with the above procedure, but starting with the 17a-bromoethynyl-19-nor-4-androstene-175-01-3- one, the 17a-chloroethynyl-19-nor-5(10)-androstene-17;8- ol-3-one, or the 17a-bromoethynyl-1-9-nor-5(10)-androstene-17B-ol-3-one in place of the 17a-chloroethynyl-19- nor-4-and-rostene-l7/8-ol-3-one, there is obtained the 21- bromo-19-nor-4-pregnene-l7B-ol-3-one, the 21-chlor0-19- nor-(10)-pregnene-17[i-ol-3-one, or the 21-bromo-19-nor- 5 -pregnene-17 3-ol-3-one respectively.

32 We claim: 1. 17a-bromoethynyl-19-nor-3,5-androstadiene-3,17B- diol diacetate.

2. 17a chloroethynyl-19-n0r-5-androstene-3fi,l7fl-diol- 5 17-acetate.

3. 17a-br0moethynyl-19-n0r-5-androstene-3fl,l7fi-diol- 17-acetate.

4. 17x-chloroethyny1-19-nor-androstane-3fl,17,8-diol- 5a,6a-oxide-17-acetate.

5. l7a-bromoethynyl-l9-nor-androstane-3fl,17,8-diol- 5a,6a-oxide-l7-acetate.

6. 6a-methyl-l7a-chloroethynyl-19-nor-4-androstene- 17fl-methoXy-3-one.

7. 6a-methyl-17a-bromoethyny1-l9-nor-4-androstene- 175-methoxy-3-one.

8. 6a-methyl-17a-chloroethynyl-l9-nor-4-androstene- 17fl-ol-3-one acetate.

9. 6a-methyl-17a-bromoethynyl-19-n0r-4-andr0stene- 17fi-ol-3-one acetate.

10. A compound of the following formula OR I j' -czo c1 wherein R is an acetyl radical.

N 0 references cited.

35 LEWIS GOTTS, Primary Examiner.

L. H. GASTON, MORRIS LIEBMAN, Examiners. 

4. 17A-CHLOROETHYNYL-19-NOR-ANDROSTANE-3B,17B-DIOL5A,6A-OXIDE-17-ACETATE. 